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This report contains the collective views of an international group of experts anddoes not necessarily represent the decisions or the stated policy of the World Health Organization

WHO Technical Report Series927

WHO EXPERT COMMITTEEON BIOLOGICAL

STANDARDIZATION

Fifty-fourth report

World Health OrganizationGeneva 2005

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WHO Library Cataloguing-in-Publication Data

WHO Expert Committee on Biological Standardization (2003 : Geneva, Switzerland)WHO Expert Committee on Biological Standardization : fifty-fourth report.

(WHO technical report series ; 927)

1.Biological products — standards 2.Vaccines — standards 3.Blood4.Reference standards 5.Guidelines I.Title II.Series

ISBN 92 4 120927 5 (LC/NLM classification: QW 800)ISSN 0512-3054

WHO Expert Committee on Biological Standardization

© World Health Organization 2005

All rights reserved. Publications of the World Health Organization can be obtained from Marketing andDissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22791 2476; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce ortranslate WHO publications — whether for sale or for noncommercial distribution — should be addressedto Publications, at the above address (fax: +41 22 791 4806; e-mail: permissions@who.int).

The designations employed and the presentation of the material in this publication do not imply theexpression of any opinion whatsoever on the part of the World Health Organization concerning the legalstatus of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiersor boundaries. Dotted lines on maps represent approximate border lines for which there may not yet befull agreement.

The mention of specific companies or of certain manufacturers’ products does not imply that they areendorsed or recommended by the World Health Organization in preference to others of a similar naturethat are not mentioned. Errors and omissions excepted, the names of proprietary products are distin-guished by initial capital letters.

The World Health Organization does not warrant that the information contained in this publication iscomplete and correct and shall not be liable for any damages incurred as a result of its use.

This publication contains the collective views of an international group of experts and does not necessarilyrepresent the decisions or the stated policy of the World Health Organization.

Typeset in Hong KongPrinted in Singapore

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Contents

Introduction 1

General 1Developments in biological standardization 1Progress reports and work programmes 5Contamination of oral poliomyelitis vaccine with wild poliovirus 7Feedback from the field on WHO recommendations and guidelines

and capacity building in quality assurance 8

International guidelines, recommendations and other matters related to themanufacture and quality control of biologicals 9

Guidelines on nonclinical evaluation of vaccines 9Recommendations for the production and control of pneumococcal

conjugate vaccines 9Recommendations for the production and control of influenza

vaccine (inactivated) 10Requirements for the use of animal cells as in vitro substrates for

the production of biologicals 11Requirements for diphtheria, tetanus and pertussis and combined

vaccines 11Potency assays for acellular pertussis vaccines 12Requirements for the collection, processing and quality control of

material for transplantation 13Human and animal spongiform encephalopathies and safety of

biologicals 13Gene transfer medical products 14Proposals for discontinuation of international requirements and

guidelines 14

International reference materials 15Proposal for discontinuation of reference materials 15Recommendations for the preparation, characterization and

establishment of international and other biological reference materials 15

Antibodies 17Anti-toxoplasma serum, human 17

Antigens and related substances 17Smallpox reference materials 17Yellow fever vaccine 18Pertussis toxin 19Diphtheria and tetanus reference materials 19

Blood products and related substances 20Factor VIII, concentrate 20Factor VIII/von Willebrand factor, plasma 21Low-molecular-weight heparin 21Prekallikrein activator 22

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Cytokines, growth factors and endocrinological substances 22WHO consultation on cytokines, growth factors and endocrinological

substances 22Human interferon beta 23Tumour necrosis factor alpha, human 24Luteinizing hormone, recombinant 24Thyroid-simulating hormone for immunoassay 25Interferon neutralizing antibody tests 26

Diagnostic reagents 26WHO consultation on international standards for testing diagnostic

kits used in detection of hepatitis B surface antigen andanti-hepatitis C virus antibodies 26

Hepatitis B surface antigen 27Lipoprotein (a) 29

Miscellaneous 29Standardization of human papilloma virus vaccine 29Standardization of human immunodeficiency virus neutralizing

antibody assays 30

Annex 1WHO guidelines on nonclinical evaluation of vaccines 31

Annex 2Recommendations for the production and control of pneumococcalconjugate vaccines 64

Annex 3Recommendations for the production and control of influenza vaccines(inactivated) 99

Annex 4Requirements for the use of animal cells as in vitro substrates forthe production of biologicals (Addendum 2003) 135

Annex 5Recommendations for diphtheria, tetanus, pertussis and combinedvaccines (Amendments 2003) 138

Annex 6Biological substances: international standards and reference reagents 148

Annex 7Recommendations and guidelines for biological substances usedin medicine and other documents 151

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WHO Expert Committee on Biological StandardizationGeneva, 17–21 November 2003

MembersDr W.G. van Aken, Amstelveen, the Netherlands (Chairman)

Dr D.H. Calam, Pewsey, Wiltshire, England (Rapporteur)

Dr M. de los Angeles Cortes Castillo, Director, Quality Control, National Instituteof Hygiene, Mexico City, Mexico

Dr R. Dobbelaer, Head, Biological Standardization, Scientific Institute of PublicHealth, Brussels, Belgium (Vice-Chairman)

Dr F. Fuchs, Director, Lyon Site, French Agency for Safety of Health Products,Lyon, France

Dr B. Kaligis, International Relations Division Head, Bio Farma, Bandung,Indonesia

Dr N.V. Medunitsin, Director, Tarasevic State Institute for the Standardizationand Control of Medical Biological Preparations, Moscow, Russian Federation

Dr F. Ofosu, Department of Pathology and Molecular Medicine, McMasterUniversity, Hamilton, Ontario, Canada

Dr F. Reigel, Head, Biological Medicines and Laboratories, Swissmedic, Agencyfor Therapeutic Products, Berne, Switzerland

Representatives of other organizations

Developing Country Vaccine Manufacturer’s NetworkDr S. Jadhav, Executive Director (Quality Assurance), Serum Institute of India

Ltd., Maharashtra, Pune, India

European Diagnostic Manufacturers AssociationDr J. Diment, Scientific and Technical Manager, North Europe, Asia Pacific and

Export, Ortho-Clinical Diagnostics, Amersham, England

Council of Europe, European Directorate for the Quality of MedicinesMr J.-M. Spieser, Head, Division IV Biological Standardisation/Network of Official

Medicines Control Laboratories (OMCLs), Strasbourg, France

International Association of BiologicalsDr A. Eshkol, Senior Scientific Adviser, Aeres Serono International SA, Geneva,

Switzerland

International Federation of Clinical Chemistry and Laboratory MedicineProfessor J. Thijssen, c/o Department of Endocrinology, University Hospital

Utrecht, Utrecht, the Netherlands

International Federation of Pharmaceutical Manufacturers AssociationsDr M. Duchêne, Director, Quality Control, GlaxoSmithKline Biologicals,

Rixensart, Belgium

Dr A. Sabouraud, Director, Quality Control of Development Products, AventisPasteur S.A., Marcy l’Etoile, France

International Society on Thrombosis and HaemostasisProfessor I.R. Peake, Division of Genomic Medicine, Royal Hallamshire Hospital,

Sheffield, England

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Plasma Protein Therapeutics AssociationDr E. Hutt, Brussels, Belgium

United States PharmacopeiaDr T. Morris, Department of Information and Standards Development, Rockville,

MD, USA

SecretariatDr T. Barrowcliffe, Head, Division of Haematology, National Institute for Biological

Standards and Control, Potters Bar, Herts., England (Temporary Adviser)

Dr A. Bristow, Head, Division of Endocrinology, National Institute for BiologicalStandards and Control, Potters Bar, Herts., England (Temporary Adviser)

Dr M. Corbel, Head, Division of Bacteriology, Division of Bacteriology, NationalInstitute for Biological Standards and Control, Potters Bar, Herts., England(Temporary Adviser)

Dr R. Decker, Hepatitis and AIDS Research, Deerfield, Illinois, USA (TemporaryAdviser)

Dr W. Egan, Acting Director, Office of Vaccines, Center for Biologics, Evaluationand Research, Food and Drug Administration, Rockville, MD, USA(Temporary Adviser)

Dr J. Epstein, Director, Office of Blood Research and Review, Center forBiologics Evaluation and Research, Food and Drug Administration, Rockville,MD, USA (Temporary Adviser)

Dr I. Feavers, Division of Bacteriology, National Institute for Biological Standardsand Control, Potters Bar, Herts., England (Temporary Adviser)

Dr E. Griffiths, Associate Director General, Biologics and Genetic Therapies,Ottawa, Ontario, Canada (Temporary Adviser)

Dr M.F. Gruber, Scientific Reviewer, Division of Vaccines and Related ProductsApplication, Center for Biologics Evaluation and Research, Food and DrugAdministration, Rockville, MD, USA (Temporary Adviser)

Dr S. Inglis, Director, National Institute for Biological Standards and Control,Potters Bar, Herts., England (Temporary Adviser)

Dr L. Jodar, Associate Director for Programmes, Institutional Development andPartnerships, International Vaccine Institute, Kwanak-Ku, Seoul, Republic ofKorea (Temporary Adviser)

Dr N. Lelie, Viral Quality Control Unit, Sanquin-CLB, Alkmaar, the Netherlands(Temporary Adviser)

Dr P.D. Minor, Head, Division of Virology, National Institute for BiologicalStandards and Control, Potters Bar, Herts., England (Temporary Adviser)

Dr M. Nübling, Paul Ehrlich Institute, Langen, Germany (Temporary Adviser)

Dr J. Wood, Division of Virology, National Institute for Biological Standards andControl, Potters Bar, Herts., England (Temporary Adviser)

Dr D. Wood, Coordinator, Quality Assurance and Safety of Biologicals, WorldHealth Organization, Geneva, Switzerland (Secretary)

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Introduction

The WHO Expert Committee on Biological Standardization metin Geneva from 17 to 21 November 2003. The meeting was openedby Dr Vladimir Lepakhin, Assistant Director-General, HealthTechnology and Pharmaceuticals, WHO, on behalf of the Director-General.

Dr Lepakhin emphasized the importance to Member States of thework of the Committee in preparing recommendations for biologicalproducts and for the development, establishment and distribution ofreference materials for biologicals. During the meeting, a number ofproposals for reference materials and draft recommendations wouldbe considered. Dr Lepakhin informed the Committee that the WorldHealth Assembly had endorsed many resolutions on the subjects ofquality, safety and efficacy of medicines, blood products, vaccines andother biologicals. Despite considerable progress made both by WHOand its Member States in the implementation of such directives,urgent action was needed to sustain and expand basic normativeregulatory functions underpinning public health efforts to assure ac-cess to quality biological medicines. Furthermore, as a result of rap-idly changing global environments, increased international trade andthe opening of borders, many biological medicines, including bloodproducts, were circulating more freely than ever. Unless they weresubject to control, biological medicines such as those derived fromblood and plasma could be vehicles for the transmission of infectiousdiseases and/or other emerging agents. Dr Lepakhin reminded theCommittee that the establishment and functioning of national regula-tory systems with reference to WHO recommendations, norms andstandards was essential to protect patients and public health fromfraudulent practices and economic waste. WHO was therefore seek-ing political commitment and support from Member States to sustainand increase capacity building and training in all aspects of regulatoryfunctions, including the efficient implementation of good regulatorypractices. Finally, Dr Lepakhin reminded the Committee that its de-cisions should be based on sound science and common sense and noton partisan considerations.

General

Developments in biological standardization

The Committee was informed of the organizational changes that hadtaken place at WHO headquarters. The two teams concerned with

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biological standardization work on an integrated programme al-though based in different clusters. The outcomes of the standardiza-tion programme include recommendations for quality standards,provision of reference materials, development of consensus onquality-related issues for biologicals and building technical capacityfor biologicals in the different WHO Regions. The role of the Interna-tional Laboratories in the development of this programme was ac-knowledged. The plans for further development of laboratorysupport for biological standardization by broadening geographicalrepresentation and improving working relationships with existinglaboratories through regular meetings and better definition of priori-ties were outlined. A review of the Expert Advisory Panel on Biologi-cal Standardization had shown the need for more even geographicaland gender representations and for the identification of more expertscurrently working the field. The need to enhance support for theExpert Committee had also been recognized and this could usefullybe addressed through the greater use of Working Groups to prepareand review proposals for reference materials and recommendations.Consideration was also being given to how the work and operation ofthe Expert Committee might evolve to meet future needs in a moretimely and efficient manner.

The place of standardization of vaccines within the Immunization,Vaccines and Biologicals department was outlined. Establishment ofnorms and standards is a critical early stage in the development ofnew vaccines. There is increasing appreciation by users of the normsand standards of the impact of standardization on global public healthand trade, leading to increased support, both in terms of financial andhuman resources, and also to increased demand for standardizationactivities. These demands are likely to require innovative ways ofproviding support. As an example, the generous support of theGovernment of the Republic of Korea, through staff secondmentfrom the Korean Food and Drug Administration, was acknowledged.The priorities for vaccine standardization for the next 2 years hadbeen identified as: the provision of written guidelines and referencematerials for new vaccines; improving the scientific basis for establish-ing vaccine quality; new and updated WHO recommendations onproduction and quality control; selected international reference mate-rials; improved global coordination of standardization and control forvaccines; continued contributions to the safety of vaccines throughguidelines on production and quality control; and polio vaccine stan-dards for use after WHO has certified the global eradication of polio.These priorities are being addressed through the establishment of agroup to discuss quality working through a controlled-access web site

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and teleconferencing; better prioritization of recommendations beingconsidered by the Expert Committee; production of updated guide-lines for production of international biological reference materialsand development of regional reference materials; and an initiative toestablish a WHO repository for vaccine seeds of global importance topublic health.

Attention was drawn once again to the unacceptable delays inpublication of the formal reports of Expert Committee meetings. TheCommittee noted that such delays had persisted despite their previ-ous recommendations to address the issue. The Committee was seri-ously concerned about the consequences of such delays particularlyfor regulatory authorities who are unable to implement unpublishedrecommendations. Pre-publication of the draft report and its annexesdoes not provide an adequate substitute for the published recommen-dations. The Committee recommended that WHO gives urgent atten-tion to seeking ways to overcome the delays and so ensure that thevalue of work on biological standardization is not lost.

Finally, the Committee was updated on several topics not consideredelsewhere during the meeting. The Committee was informed of aninformal WHO consultation held recently on the development ofa vaccine for severe acute respiratory syndrome (SARS). Severalapproaches are under investigation and it is expected that trials ofcandidate inactivated SARS coronavirus vaccines may start shortly.In this context it was noted that WHO had recommended that allwork with live SARS coronavirus, including the large-scale produc-tion of virus during the initial stages of production of inactivatedvaccine, should be performed under biosafety level 3 conditions.Work is under way to identify validated animal models for efficacyand safety. The safety model should address concerns about immuneenhancement, a phenomenon seen with one inactivated animalcoronavirus vaccine. WHO would facilitate the continuation of workon quality and safety issues. Studies should be initiated on establish-ing antibody standards for the calibration of assays and diagnostictests, and for the calibration of immunotherapy products.

The Committee was informed of new initiatives within WHO thatwould have an impact on activities in the area of blood products andrelated biologicals during the next biennium, 2004–2005. This area,which is part of the overall biologicals programme, is placed withinthe new Department of Essential Health Technologies which, in turn,is part of the Health Technology and Pharmaceuticals cluster.The new initiatives are centred around a strong commitment tocapacity building of national regulatory authorities, and aimed at

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strengthening their technical capacity especially in the area of bloodproducts. Basic operational frameworks have been developed, basedon the four objectives: policy; quality and safety; access; and use.The basic operational framework is an assessment tool that will beused to identify priorities and activities at the level of WHO Regionsand countries during the next biennium, and also to assist countriesto identify gaps in their own health systems. Two countries would beselected from each WHO region to pilot-test the assessment tool. Thearea of biological standardization would be given special emphasisto highlight the need to develop regulatory mechanisms for plasmafractionation activities, either where these activities take place in thecountry already or where countries are expected to become involvedin offering contracts for plasma fractionation in the near future. Strin-gent regulatory control is vital for assuring the quality and safety ofproducts derived from human blood. Special efforts will be devoted tostrengthening the technical capacity of national regulatory authoritiesto ensure the appropriate control of blood products and related invitro diagnostics worldwide.

The priorities for 2004–2005 will include the updating of the WHOrequirements for the collection, processing and quality control ofblood, blood components and plasma derivatives. Training activitiesfor good manufacturing practices at blood and plasma collectioncentres will be initiated in the Region of the Americas with thecollaboration of the Regional Office. Efforts will be made to facilitatethe availability of those international biological reference materialswith the greatest importance to public health, such as the referencepanel for hepatitis B surface antigen (HbsAg) (see p.27), in countrieswhere resources are limited with the collaboration of WHO RegionalAdvisers. Activities relating to the standardization of in vitro bio-logical diagnostic materials, particularly those applicable to testingfor virological safety, will continue.

An overall need in the area of biological standardization is for coor-dination of the process of setting international standards. To achievethis WHO, and the Expert Committee on Biological Standardization,in particular should enhance their work with regulators in the interna-tional sphere, for example through the International Conference ofDrug Regulatory Authorities as well as with other standard-settingbodies such as the International Standards Organisation and theInternational Office for Weights and Measures.

The Committee welcomed the comprehensive review of the work onbiological standardization but expressed concern that despite the im-portance of the extensive programme proposed, and its likely impact

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on public health, the relevant teams did not have sufficient personneland resources to effectively perform the proposed work. The Com-mittee recommended that these concerns be addressed.

Progress reports and work programmes

The Committee was informed of recent developments at the variousWHO International Laboratories for biological standardization.

National Institute for Biological Standards and Control,Potters Bar, England

The Committee was provided with an updated programme ofwork proposed by the National Institute for Biological Standards andControl (NIBSC) for the period 2003–2008 covering work in progress,reference materials expected to require replacement and newprojects. The Committee was pleased to note progress made with thework programme since its previous review, represented by a numberof items considered elsewhere on the Agenda. The new Centre forBiological Reference Materials at the Institute had now been opened.This facility provides increased capacity for small- and large-scale fillsof ampoules and vials, manufactured under negative pressure con-ditions to high quality standards. One filling suite can process up to20 000 ampoules in a single batch and the other is capable of process-ing up to 23 000 vials in a batch. The previous maximum batch size wasof the order of 3000 ampoules or vials. The complex and technicallyadvanced building is currently undergoing validation. The facilityrepresents a very significant investment by the United Kingdom De-partment of Health that will be of benefit to international biologicalstandardization. The Committee was also informed of some organiza-tional changes planned within the Institute to take best advantage ofthe new facility. Another new biological resource at NIBSC is theestablishment of the UK stem-cell bank intended to hold and distrib-ute cells for research purposes and for “clinical grade” cells under aregime that complies fully with regulatory requirements for qualityassurance. An independent committee will be formed to overseedecisions on distribution of cells. There will be no involvement inbasic research on stem-cell biology or in any commercial productdevelopment.

Central Laboratory of the Netherlands Red Cross Blood TransfusionService, (Sanquin), Amsterdam, the Netherlands

The Central Laboratory of the Netherlands Red Cross BloodTransfusion Service makes available reference materials for use in theblood products field. The Committee was informed about reference

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materials distributed during the past year. The Committee was alsoinformed of the need to replace two standards because of depletion ofstocks (thromboplastin, rabbit, plain and anti-double stranded DNA,serum, human) and of the development of new human serum stan-dards each monospecific for a specific hepatitis C antigen. Candidatematerials would be obtained for evaluation. Sanquin works withNIBSC in the blood products field, particularly on reference materialsfor quality control in the context of blood virology. A number ofsuch materials had been identified and work on them is in progress.Another area in which there is a need for reference materials isquantitative measurement using nucleic acid amplification techniques(NAT). The Committee noted that further information on the needfor the anti-double stranded DNA serum would be helpful in deter-mining the priority assigned to this work and endorsed the workprogramme under way at Sanquin.

c) Center for Biologics Evaluation and Research, Bethesda, MD,United States

The Committee was informed of recent developments in the vaccinefield in the United States. Two new vaccines, a nasally-administeredlive attenuated influenza vaccine and a diphtheria and tetanus vaccinefor use in adults, had been licensed during 2003. Current safety con-cerns were the implications of contamination of some vaccines morethan 40 years ago with Simian virus 40 (SV40) virus and the adequacyof past and current testing for SV40. There is strong evidence tosuggest that SV40 is a transforming virus and moderate evidence tosuggest that exposure to SV40 could lead to human cancer undernatural conditions. However, the evidence for accepting or rejecting acausal relationship between SV40-containing vaccines administeredin the 1950s and cancer is inadequate. The current concerns aboutSV40 emphasize the potential for very long-term implications of is-sues of vaccine quality. The Committee was also informed that oneconsequence of the case of bovine spongiform encephalopathy (BSE)identified in Canada in 2003 was that the current US regulationsconcerning ruminants and ruminant products from countries withmimimal risk of BSE may be revised.

The Committee was further informed of current developments in theregulation of blood products including the management of emerginginfectious diseases. There had been a rapid development of investiga-tional NAT techniques for West Nile virus in the United States fol-lowing the emergence of this virus in 2002. As a result a number ofcontaminated donations had been identified in 2003 and withdrawnfrom the blood supply. A control panel was under development. The

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implications of other infections, including monkeypox, smallpox andanthrax, on selection of blood donors were reviewed. Current policycalls for donor deferral (i.e. a delay between vaccination and clear-ance to donate blood) subsequent to smallpox vaccination because ofthe uncertainty over viraemia, and there are concerns over the impactof a mass vaccination campaign against smallpox on the blood supply.Progress in the development of diagnostic tests for screening theblood supply was described, and information given about recentguidance from the US Food and Drug Administration in the bloodproducts field.

The Committee noted the information supplied, endorsed the workprogrammes proposed by the laboratories and expressed gratitude fortheir contributions. The Committee further noted those activitiesintended to anticipate developments in standardization and con-sidered that these activities would allow time to ensure adequateresources for this work.

Contamination of oral poliomyelitis vaccine with wild poliovirus

The Committee was informed that, in the period from November2002 to February 2003, several cases of poliomyelitis had been re-ported in one country after vaccination, and the MEF-1 referencewild-type poliovirus type 2 strain was isolated in each case. Initialinvestigations excluded cross-contamination within the WHO poliolaboratory network as an explanation for the findings. Further exami-nation demonstrated the presence of MEF-1 in vials of one vaccinebatch that had been filled locally from imported bulk material.Samples of the batch that had been collected from the field, fromretained samples and from other bulks were tested. Only samplesfrom the field tested positive, which indicated that contamination hadtaken place downstream from manufacture although it could not beestablished whether this was during storage or distribution. Enhancedsecurity measures have now been put in place by the manufacturerconcerned. The Committee was asked to consider the wider implica-tions of this episode, whether current WHO guidance on good manu-facturing practice (GMP) is adequate and whether additional controlmeasures on the final product should be specified. The Committeeconsidered that the prevention of deliberate interference with a prod-uct requires safeguards different to those normally covered by GMPand that no changes to existing GMP guidance for this reason werenecessary. However, the Committee drew attention to measures thatensure that vials are tamper-proof and to procedures that are avail-able to detect counterfeiting. In this context WHO was advised to

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obtain additional specialist advice, and to make this available toall vaccine manufacturers, their distributors and national regulatoryauthorities.

Feedback from the field on WHO recommendations andguidelines and capacity building in quality assurance

The Committee was informed of feedback on the recommendationsand guidelines that it had produced on GMP. This had been obtainedin part from the field through the WHO Global Training Networkcourses on GMP organized for Indian and Chinese GMP inspectorsand quality assurance managers of vaccine manufacturers, in July2002, August 2003 and November 2003. The problems that had beenidentified included some ambiguities and some inconsistencies inwording; these were outlined for future revision. It was pointedout that GMP inspectors need to understand the background of therequirements and that proper interpretation of WHO guidelines iscritical when national guidelines are being prepared.

The Committee was reminded that the recommendations for GMPfor biologicals published in 1992 (WHO Technical Report Series No.822, 1992) apply to all biologicals. However, the special consider-ations applicable to blood products are not adequately addressed.Some of these issues are covered by the Guidelines on collection,processing and quality control of blood, blood components andplasma derivatives (WHO Technical Report Series No. 1994, 840).Some are also covered in a Pharmaceutical Inspection Convention(PIC)/Blood circle document. Application of GMP to blood collec-tion establishments is seen as a critical area for the improvement ofblood safety. Training for GMP should have high priority and stepsare being taken to improve this through courses and the establish-ment of regional networks and regulatory forums. Feedback fromthese courses should be provided to the Committee.

The Committee noted the issues that had been raised and agreed thatpriority should be given to a review of GMP for blood products.Moreover, this review should be given a higher priority than therevision of the Guidelines on collection, processing and qualitycontrol of blood, blood components and plasma derivatives (WHOTechnical Report Series No. 840, 1994).

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International guidelines, recommendations andother matters related to the manufacture andquality control of biologicals

Guidelines on nonclinical evaluation of vaccines

Because of scientific and technical developments, improvements arebeing made to existing vaccines and a broad range of novel vaccinesare under development. There is a need for guidance on the type andextent of nonclinical evaluation needed for such products, basedon best scientific knowledge, since this forms an essential part ofthe development of the vaccines. The Committee noted the draftWHO Guidelines on Nonclinical Evaluation of Vaccines (WHO/BS/03.1969), which had been prepared following the preliminary reviewof an earlier draft at the fifty-third meeting of the Committee. TheGuidelines are intended to set out principles for nonclinical evalua-tion of vaccines and to provide information and guidance to vaccinemanufacturers and recommendations for national regulatory authori-ties. The document outlines regulatory expectations and is intendedto complement, and therefore should be read in conjunction with, theGuidelines for clinical evaluation of vaccines: regulatory expectations(WHO Technical Report Series, No. 924, 2004). After making a num-ber of changes to the text, the Committee adopted the revised text asthe Guidelines on nonclinical evaluation of vaccines and agreed thatit should be annexed to its report (Annex 1).

Recommendations for the production and control ofpneumococcal conjugate vaccines

The Committee reviewed the draft WHO Recommendations for theproduction and control of pneumococcal conjugate vaccines (WHO/BS/03.1968). The Recommendations are intended to be scientific andadvisory and provide information and guidance to national regulatoryauthorities and vaccine manufacturers. Infections caused by Strepto-coccus pneumoniae, the pneumococcus, are responsible for substan-tial morbidity and mortality, particularly in the very young and in theelderly. Several pneumococcal vaccines containing polysaccharideconjugated to protein carriers are available and others are at anadvanced stage of development. Controlled clinical trials of thesevaccines have demonstrated that such conjugates are both safeand highly immunogenic. Differences in the incidence of the sero-types causing disease from one continent to another have led tothe development of pneumococcal vaccine formulations consistingof increasing numbers of conjugated components. The experience

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gained with identification of reference levels of antibodies that sup-ported the successful licensure of one product in a number of coun-tries will guide the review of clinical trial data from other countriesand with other products.

The draft was based on the outcome of an informal WHO Consulta-tion held in 2003. The Committee was reminded of the large numberof serotypes of S. pneumoniae and of the need to allow flexibilityin the recommendations to cover different conjugation chemistriesand carrier proteins. After making several changes to the text, theCommittee adopted the revised text as the Recommendations for theproduction and control of pneumococcal conjugated vaccines andagreed that it should be annexed to its report (Annex 2).

Recommendations for the production and control of influenzavaccine (inactivated)

The Committee reviewed the draft WHO recommendations for theproduction and control of influenza vaccine (inactivated) (WHO/BS/03.1967). The recommendations are intended to provide informationand guidance for manufacturers of vaccines and recommendations fornational regulatory authorities. The draft was based on the outcomeof an informal WHO Consultation held in Ferney-Voltaire, France,in July 2003, during which a previous draft had been revised. TheCommittee was reminded of the significant developments in influenzavaccines during the past years. Subunit and split vaccines are nowwidely used, and the effective dose of haemagglutinin has been estab-lished. In addition vaccines containing adjuvants had been developedand approved. The danger of pandemics caused by the appearanceof novel and highly pathogenic strains of virus presents a number ofchallenges for the production and administration of suitable vaccines.The existing recommendations therefore require revision to reflectthese and other developments. After making several changes to thetext, the Committee adopted the revised text as the Recommenda-tions for the production and control of influenza vaccine (inactivated)and agreed that it should be annexed to its report (Annex 3).The Committee noted that the recommendations from the informalConsultation included: work to standardize the virus neutralizationtest; to establish its correlates of immunity and to evaluate its use forvirus strain characterization; and to prepare vaccines through reversegenetic techniques and evaluate their use for vaccine productionsubject to resolution of intellectual property issues relating to thetechnique.

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Requirements for the use of animal cells as in vitro substrates forthe production of biologicals

The WHO requirements for the use of animal cells as in vitro sub-strates for the production of biologicals (WHO Technical ReportSeries No. 878, 1998, Annex 1) provide, inter alia, information abouta WHO cell bank of Vero cells. These cells were developed in 1987and designated as a Master Cell Bank in 1998. Cultures of the cells areavailable to manufacturers and national control authorities. As at itsfifty-third meeting (WHO Technical Report Series, No. xxx, in press),the Committee had been informed of possible deficiencies in therecords relating to the cell bank that might have regulatory implica-tions for the establishment of master cell banks, and a revision of theRequirements was therefore proposed. A draft amendment to thesection “General considerations — continuous-cell-line substrates”in the Requirements had been prepared (WHO/BS/03.1970). TheCommittee adopted the draft text as the Addendum 2003 to therequirements for the use of animal cells as in vitro substrates forthe production of biologicals and agreed that it should be annexed toits report (Annex 4).

Requirements for diphtheria, tetanus and pertussis andcombined vaccines

The Committee was informed of the developments that had takenplace in methods of assay of diphtheria and tetanus vaccines since theRequirements for diphtheria, tetanus and pertussis and combinedvaccines (WHO Technical Report Series, No. 800, 1990, Annex 2) hadbeen published. These developments had been directed to overcom-ing difficulties in potency testing and several in vitro assays had beendeveloped and validated. A series of reviews and meetings held dur-ing 1999–2000 had resulted in proposals for amendment of the WHORequirements, but the technical details could not be finalized at thattime. Further discussion resulted in a recommendation to move to aharmonized and simplified batch release assay using guinea-pigs. TheCommittee noted draft amendments to the WHO requirements fordiphtheria, tetanus and pertussis and combined vaccines (WHO/BS/03.1984). The draft was based on the outcome of an informal meetingof a WHO Expert Group held in Geneva from 30 June to 2 July 2003.The main changes to the present Requirements constitute an updat-ing of the sections on reference materials, and splitting sections onpotency into two addressing licensing and batch release, respectively.After making a number of changes to the draft, the Committeeadopted the modified text as the Amendments 2003 to the Require-ments for diphtheria, tetanus and pertussis and combined vaccines

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(WHO Technical Report Series, No. 800, 1990, Annex 2) and agreedthat it should be annexed to its report (Annex 5).

Potency assays for acellular pertussis vaccines

The Committee was informed of the meeting of a WHO WorkingGroup on standardization and control of pertussis vaccines held inFerney-Voltaire, France, in May 2003. The purpose of the meetingwas to advise WHO on updating the guidelines for the acellularpertussis component of monovalent or combined vaccines. Previousmeetings had resulted in the initiation of collaborative studies toassess the value of intranasal challenge assays and modified intracere-bral challenge assays. Data from the studies were reviewed at themeeting. One study showed that the intranasal assay was transferablebetween laboratories and differentiated between the collaborativestudy samples, but needed to be optimized to allow estimates ofrelative potencies of products. A reference preparation included inthe study had performed satisfactorily, but may not be an ideal refer-ence for all types of acellular pertussis vaccines. A second studyevaluated a modified intracerebral assay. This was found to be effec-tive for assigning relative potencies to acellular pertussis vaccinesalthough it was noted that active pertussis toxin increased the appar-ent potency of preparations in the assay. A reference material, JNIH-3, included in the study had proved satisfactory.

The modified intracerebral challenge test is used in some parts of theworld whereas an immunogenicity assay is used in other countries forlot release. Although it was acknowledged that it would be difficult tointroduce changes for products and countries that had effectivelymonitored acellular pertussis vaccines for more than 20 years, it wasagreed that additional methods for the assessment of functional activ-ity for new products and formulations, and for technology transfer,were needed. The Committee noted that the Working Group hadrecommended that position papers about several assays and testsshould be prepared by small subgroups for eventual submission to theCommittee. The Working Group also recommended that the currentWHO Recommendations for whole cell pertussis vaccines should beupdated; that steps should be taken to make available an interna-tional reference antiserum for pertussis antigens before the currentlywidely used Center for Biologics Evaluation and Research (CBER)material runs out; and that NIBSC should be asked to confirm thatsupplies of monoclonal antibodies to Fim 2 and Fim 3 for use asserotyping agents are adequate. The Committee endorsed theserecommendations.

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Requirements for the collection, processing and quality control ofmaterial for transplantation

The Committee was informed of the growing numbers of transplantsinvolving organs, tissues and cells worldwide. Limitations of supplydetermine the number of transplants that can be performed and pre-vent demand being met. There is a wide variety of tissue banks, andcross-boundary circulation of material takes place within and be-tween all WHO Regions. Complex processes may be required forhandling tissues and cells for transplantation. Ethical and publichealth concerns arise with respect to use of human tissue, the risk oftransmission of pathogens and misuse of resources. These concernswere addressed at a WHO meeting in Madrid, Spain, in October 2003which recommended that WHO develop quality, technical and ethicalstandards for transplantation. These should address safety and effi-cacy, GMP and quality management systems, and be consistent withWHO recommendations for other therapeutic materials. The Com-mittee was invited to participate in this work. Although this repre-sents a new sphere of activity, the Committee agreed that it had arole to play and asked for detailed and prioritized proposals to besubmitted to it.

Human and animal spongiform encephalopathies and safetyof biologicals

The Committee was reminded that an informal WHO Consultationon transmissible spongiform encephalopathies (TSEs) in relation tobiological and pharmaceutical products had been held in Geneva inFebruary 2003. The objectives of the Consultation were to provideevidence-based information to regulatory authorities, especially incountries where BSE had not yet been reported, regarding the riskassessment and precautionary and control measures for biologicaland pharmaceutical products. An additional goal was to promoteworldwide harmonization of the regulations concerning TSE. Up-dated scientific and geographical information was presented at theConsultation and a new tissue classification was agreed. This tissueclassification was now being used as the basis of worldwide regula-tions. At its previous meeting, the Committee had requested to seethe report so that the implications for biological standardization couldbe considered. The Committee noted the report (WHO/BCT/QSD/03.01) and requested that this document be referenced where appro-priate in WHO Recommendations and Guidelines for the productionand quality control of biological medicines.

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Gene transfer medicinal products

The Committee was informed that the WHO Gene Transfer Monitor-ing Group had met in Geneva in June 2003. During the meeting thelessons learned from the adverse events that had been reported ingene therapy trials were considered together with experience gainedin preclinical tests and clinical trials; activities of the WHO WorkingGroup on Standardization and Control of Nucleic Acid Vaccines; thecurrent status of regulations and guidance; and standardization andnomenclature of gene transfer medicinal products. A draft report ofthe information presented and discussions held at the meeting waspresented to the Committee. The Monitoring Group recommendedthat WHO should develop guidelines on gene therapy to addressmanufacture of clinical grade gene therapy products and the initiationand conduct of clinical trials. These guidelines should take account ofexisting guidance. The Committee also noted that discussions arecontinuing on reference materials for gene transfer medicinal prod-ucts but, at present, there is no consensus on the most appropriateapproach to standardizing these vectors. Their diversity may evenpreclude the development of generic reference materials. The Com-mittee endorsed the proposals, noted the information that had beenpresented, and requested the Secretariat to keep it informed of fur-ther developments.

Proposals for discontinuation of international requirementsand guidelines

The Committee was informed that the Procedure for approval byWHO of yellow fever vaccines in connection with the issue of in-ternational vaccination certificates (Procedure for evaluating the ac-ceptability in principle of vaccines proposed to United Nationsagencies for use in immunization programmes (WHO TechnicalReport Series, No. 786, 1989, Annex 1)) had been superseded by theprocedure established for the pre-qualification of vaccines for supply,in principle, to United Nations agencies (WHO Technical ReportSeries No. 786, 1989). The Committee, therefore, discontinuedthe Procedure for approval by WHO of yellow fever vaccines inconnection with the issue of international vaccination certificates(1981).

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International reference materials

Proposals for discontinuation of reference materials

Apart from automatic replacement of reference materials supersededby those adopted during the meeting, the Committee was informedthat no other reference materials had been proposed fordiscontinuation.

Recommendations for the preparation, characterizationand establishment of international and other biologicalreference materials

The Committee was informed that an informal WHO Consultationhad been held in Geneva in June 2003, to consider a proposed draftrevision of the current Guidelines for the preparation, characteri-zation and establishment of international and other standards andreference reagents for biological substances (WHO Technical ReportSeries No. 800, 1990). The discussion at the meeting had identified anumber of fundamental scientific issues that would require clarifica-tion at a further Consultation.

In the establishment of WHO biological reference materials, no re-striction has usually been placed on the methods employed in thecollaborative study. This is in contrast to established practice in othermetrological fields in which a single reference method is used. TheCommittee affirmed that the methods chosen should take into ac-count the likely use of a given preparation. Where a broad range ofuses can be foreseen, it is important to continue to employ a range ofmethods in collaborative studies. This decision on appropriate meth-ods should be made clearly at the outset of the study. It should alsobe kept in mind that complex biological materials possess multi-dimensional properties, which are detected and measured in differentways by different methods that may change with time. The referencemethod approach therefore has, at best, limited applicability to bio-logical reference materials.

The second issue is the choice of units to be assigned to a particularreference preparation. Other standard-setting bodies such as theInternational Organization for Standardization (ISO) advocate theuse of Système international d’Unités (SI) units as having the highestmetrological status. However, biological reference materials are oftenof complex and unknown composition, and cannot be defined purelyin physical and chemical terms or in SI units. For this reason, theactivity or potency of such a reference material is determined bybiological procedures and stated in arbitrary International Units.The Committee considered that the choice of unit should reflect, and

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be based on, the biological and medical as well as the physicochemicalinformation available in each case.

The third and most far-reaching issue is the view of ISO that theuncertainty of the value given to a reference material should bestated. At issue, therefore, is whether, and if so how, uncertainty canbe addressed for biological reference materials. Since an arbitraryunitage is assigned to a first WHO standard, no uncertainty is associ-ated with the value. Although, in principle, an uncertainty might beestimated for replacement standards, the overriding concern of WHOis to ensure continuity of the International Unit. For reference mate-rials intended for calibration of analyses of therapeutic and prophy-lactic products, no statement about uncertainty of an assigned valuewill be made, consistent with the ISO Guide 35 which states explicitlythat reference materials in the pharmacopoeial, and by extensionmedical regulatory, context have contents stated without any uncer-tainty because of the circumstances of their use. For reference mate-rials intended for in vitro diagnostics the Committee noted that theEuropean Directive on in vitro diagnostics brings the requirements ofISO 17511 into legal effect. This would require a statement of uncer-tainty. The Committee was concerned about the scientific basis ofattaching an uncertainty to an assigned value for a biological refer-ence material and agreed that further discussions were required. Forthis purpose, the Committee was informed that several meetings wereplanned during 2004 including one with ISO and one with regulatorsconcerned with implementation of the European Directive on in vitrodiagnostic devices.

The Committee concluded that the current convention of not statingan uncertainty to the value assigned to a WHO biological referencematerial would remain unchanged. Nevertheless, they also recom-mended that memoranda accompanying reference materials estab-lished during this meeting and in future should contain a statement ofthe coefficient of variation (CV) of fill of the preparation concerned.This is one factor contributing to uncertainty. The Committee alsonoted that information about losses on storage is available to usersat the time that reference materials are formally established. TheCommittee supported a proposal to draft additional guidance withregard to stability testing of reference materials.

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Antibodies

Anti-toxoplasma serum, human

The Committee was informed that the degree of sensitivity in quanti-fying and monitoring the immunoglobulin G (IgG) response associ-ated with acute toxoplasmosis is important in supporting appropriateclinical management and that the existing standards are not suitablefor calibration of assays to distinguish between background and diag-nostic levels of IgG. The Committee noted a proposal to establisha replacement International Standard for anti-toxoplasma serum,human (WHO/BS/03.1971), based on a collaborative study per-formed by 24 laboratories in 17 countries. The study had been de-signed to assess the suitability of the candidate preparation for use incell-killing assays and to calibrate it in terms of the current Interna-tional Standard, to confirm continuity of unitage, and to assess thereactivity of the candidate in various assays. The candidate prepara-tion appeared to be stable at storage temperatures up to 20°C.The Committee noted that the value assignment results obtained inthe study were based on the currently accepted gold standard method,the dye test, and that the composition of the material means thatit is cannot be a direct replacement for the existing anti-toxoplasmastandard. In view of these considerations and on the basis of theresults obtained, the Committee established the preparation, inampoules coded 01/600, as the First International Standard forAnti-Toxoplasma IgG, Human, and assigned a potency of 20IU perampoule to it.

The Committee noted that it would be useful during the currentrevision of the Recommendations for Preparation, Characterizationand Establishment of International and other Biological ReferenceMaterials to include guidance on the maximum tolerable intra- andinter-laboratory variation during evaluation of results of collabora-tive studies.

Antigens and related substances

Smallpox reference materials

The Committee was informed that smallpox had been eradicated in1980, but that there had been a recent revival of interest in smallpoxvaccines because of the potential implications of bioterrorism activi-ties. It was considered necessary to review the existing standards andto consider whether new standards were required. Consequently acollaborative study had been performed by 13 laboratories in 10

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countries to assess the relative sensitivity of cell culture assays andchorioallantoic membrane (CAM) egg assays for smallpox vaccinesand evaluate the suitability of candidates as a replacement for thecurrent International Reference Preparation (WHO/BS/03.1977).The outcomes of the study were that, overall, there were no substan-tial differences noted in the sensitivity of the assay methods, althoughsome samples performed better in the CAM assays than in the cellculture assays and vice versa. Two suitable candidate preparationshave been identified for use as replacements. However, consistentwith the proposal from the study participants to continue to use thecurrent reference preparation, the Committee noted that the currentInternational Reference Preparation still has acceptable potency andthe existing stocks of this preparation are sufficient for the time beingso that its replacement is not urgent. The Committee thus agreed todefer any decision about replacement pending generation of furtherinformation, including stability data and information about rate ofsupply.

Yellow fever vaccine

The Committee was informed that potency determination of yellowfever vaccines has been based historically on mouse LD50 assaysalthough in vitro plaque assays have been available and in routineuse for some years. The Committee was also informed that the WHORequirements for yellow fever vaccine (WHO TRS 872, 1998, Annex2) require determination of a relationship between mouse LD50 unitsand plaque forming units (pfu) in each laboratory. The Committeenoted that this relationship is, in practice, often based on valuesestablished many years previously and that checks on the continuedvalidity of the relationship may be done infrequently. Moreover, twoor more laboratories (e.g. a manufacturer and a national controllaboratory) may assay the same batch of vaccine, but apply differentconversion factors if the relationship between mouse LD50 and pfuhad been determined at different times. The standardization of yellowfever potency determinations would benefit from an internationallyavailable reference material.

The Committee noted the report of a collaborative study performedby 13 laboratories in eight countries, intended to assess the suitabilityof candidate preparations for an international standard and the rela-tionship between the two assay methods (WHO/BS/03.1985 Rev.1).One candidate preparation appeared to be suitable for use in plaqueassays. The stability of the preparation had been studied over periodsof up to 3 years and appeared satisfactory. On the basis of the resultsof the collaborative study, the Committee established the prepara-

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tion, in ampoules coded 99/616, as the First International Standard forYellow Fever Vaccine and assigned an activity of 104.5 IU per ampouleto it. The data obtained in the study indicated that there was a consis-tent relationship between mouse LD50 and plaque assays. The Com-mittee therefore supported a proposal to encourage manufacturersand control laboratories to include the standard in assays to evaluateits suitability for setting a minimum potency of 104.0 IU for yellowfever vaccines. Data should be collated by WHO and analysed todetermine whether the potency specification given in the WHORecommendations (WHO Technical Report Series, No. 872, 1998)should be amended.

Pertussis toxin

The Committee was informed that reference preparations of pertussistoxin are required for the quality control and assessment ofpertussis vaccines. Two methods are currently used to assay residualpertussis toxin in both acellular and whole cell pertussis vaccines. TheCommittee noted a proposal to establish an international standard forpertussis toxin and the report (WHO/BS/03.1978) of a collaborativestudy performed by six laboratories in six countries using both thehistamine sensitizing and Chinese hamster ovary cell assay methods.Although the candidate preparation had been filled some years ago,its stability had been demonstrated by studies following a period ofstorage at elevated temperatures. On the basis of the results of thecollaborative study, the Committee established the preparation, inampoules coded JNIH-5, as the First International Standard forPertussis Toxin and assigned an activity of 10000IU per ampoule toit. Nominally one IU corresponds to 1 nanogram of protein nitrogenusing a conversion factor of 10.

Diphtheria and tetanus reference materials

The Committee was informed that stocks of a number of referencematerials for testing diphtheria and tetanus components in vaccineswere depleted and that several new reference materials were requiredfor use with methods that would replace tests using animals. TheCommittee was also informed of the proposed priorities for thereplacement and establishment of these materials and other studieson methodology. The Committee recommended that the followingstudies should be initiated:

— collaborative studies on candidate replacement preparationsfor use in flocculation assays for diphtheria and tetanustoxoids and on comparison of methods to measure antigenicpurity;

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— collaborative studies on a candidate replacement for the currentInternational Standard for Diphtheria Toxoid, Adsorbed; and

— collaborative studies intended to establish new WHO standardsfor diphtheria and tetanus toxins for use in cell culture assays andfor human IgG diphtheria antitoxin (for which a candidate prepa-ration is available).

The Committee noted a proposal for collaborative studies onmonoclonal antibody panels for diphtheria and tetanus toxoids foruse in ELISA assays to monitor product consistency. Although theproject was welcomed, the Committee expressed concern aboutwhether sufficient monoclonal material could be made available forlong-term use and asked for a more detailed project proposal tobe submitted.

Blood products sand related substances

Factor VIII, concentrate

The Committee was informed that stocks of the current (recombi-nant) standard were likely to be exhausted within 12 months. TheCommittee was also informed that there had been reports of difficul-ties in using recombinant material in assaying plasma-derived concen-trates. For these reasons and after wide consultation, a decision wastaken to use plasma-derived factor VIII as the replacement material.The Committee noted a proposal to establish a replacement Interna-tional Standard for Factor VIII, Concentrate (WHO/BS/03.1973),based on a collaborative study performed by 38 laboratories in 21countries in which a candidate material was compared with fourexisting reference materials for factor VIII (Fifth and Sixth Interna-tional Standards, USFDA lot 1, EPBRP batch 2). This study wasperformed in conjunction with CBER and the European Departmentfor the Quality of Medicine (EDQM) with the aim of establishing acommon batch of the reference preparation. The participants em-ployed their customary in-house assays which were all either one-stage or chromogenic methods. There were no significant differencesin the mean potency obtained by the two methods. The preparationshowed adequate stability over 2 years. On the basis of the resultsobtained, the Committee established the preparation, in ampoulescoded 99/678, as the Seventh International Standard for Factor VIII,Plasma-derived, Concentrate and assigned an activity to it of 11.0IUper ampoule. This value is considered to ensure continuity of valuesof the International Unit for different preparations in different assaysystems.

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Factor VIII/von Willebrand factor, plasma

The Committee was informed that stocks of the Fourth InternationalStandard for Factor VIII/von Willebrand Factor, Plasma were almostexhausted. This standard is important for the calibration of bothcommercial and non-commercial secondary working standards. TheCommittee noted a proposal to establish a replacement InternationalStandard for Factor VIII/von Willebrand Factor, Plasma (WHO/BS/03.1972), based on the collaborative study performed by 37 laborato-ries in 13 countries. The candidate preparation showed adequatestability over approximately 1 year. On the basis of the resultsobtained, the Committee established the preparation, in ampoulescoded 02/150, as the Fifth International Standard for Factor VIII/vonWillebrand Factor, Plasma and assigned activities to it of 0.68IU/ampoule for Factor VIII:C; 0.94IU/ampoule for Factor VIII:antigen;0.91 IU/ampoule for VWF:antigen; 0.78IU/ampoule for VWF:risto-cetin cofactor; and 0.94IU/ampoule for VWF:collagen binding. TheCommittee noted that other studies are in progress that may provideinformation on the relative clinical values of the different assays forvon Willebrand Factor that are performed.

Low-molecular-weight heparin

The Committee was informed that stocks of the current InternationalStandard for low-molecular-weight heparin were almost exhausted.The Committee was also informed that this standard is used in thedetermination of potency of low-molecular-weight heparin. TheCommittee noted a proposal to establish a replacement InternationalStandard for low-molecular-weight heparin (WHO/BS/03.1986),based on a collaborative study performed by 30 laboratories in 14countries, in which both anti-Xa and anti-IIa assays had been em-ployed. Two candidate preparations had performed similarly and thatwith the lower inter-laboratory variability had been selected. TheCommittee was informed that this preparation had shown no degra-dation over 12 months and that the stability study would continue inreal time. On the basis of the results obtained, the Committee estab-lished the preparation, in ampoules coded 01/608, as the Second Inter-national Standard for Low-molecular-weight heparin and assignedto it activities of 1097IU of anti-Xa per ampoule and 326 IU of anti-IIa per ampoule. The Committee noted that the study had beenperformed in conjunction with the EDQM with the aim of establish-ing replacement batches of European Pharmacopoeia referencepreparations.

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Prekallikrein activator

The Committee was informed that stocks of the current InternationalStandard for prekallikrein activator were almost depleted. The Com-mittee was also informed that this standard is used in the determina-tion of the level of prekallikrein activator, an impurity present inpreparations of therapeutic blood products, such as albumin. TheCommittee noted a proposal to establish a replacement InternationalStandard for Prekallikrein Activator (WHO/BS/03.1974 + Add.1),based on a collaborative study performed by 31 laboratories in 17countries using similar kinetic assays employing a variety of sub-strates. This study had been carried out in conjunction with theEDQM. The performance of the candidate preparation was satisfac-tory. The Committee noted that the preparation had shown adequatestability over approximately 1 year. On the basis of the results ob-tained, the Committee established the preparation, in ampoulescoded 02/168, as the Second International Standard for PrekallikreinActivator and assigned an activity of 29IU per ampoule to it.

Cytokines, growth factors andendocrinological substances

WHO consultation on cytokines, growth factors andendocrinological substances

The Committee was informed that a WHO informal consultation oncytokines, growth factors and endocrinological substances had beenheld in Potters Bar, England, in October 2003. The attention of theCommittee was drawn to the increasing difficulties with nomencla-ture. An extensive range of titles had been given to reference ma-terials established by WHO and a number of confusions had arisen.The Committee agreed to a recommendation that a comprehensivereview of issues of nomenclature should be carried out. The Com-mittee was also informed that the formulation of a policy concerningstability testing of reference materials after their establishment isdesirable.

Concerns have been raised relating to the development of unwantedantibodies against some therapeutic biologicals, in addition to inter-feron alfa or beta (see p.23). The availability of reference humanserum preparations containing defined and characterized antibodiesagainst such biologicals was recognized as a potentially valuable re-source. The Committee endorsed the provision of such reagentsbased on prioritization according to need and significance for publichealth.

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A work programme for cytokines, growth factors and endocrinologi-cal substances had been agreed and potential new areas of work hadbeen identified. The Committee noted the programme and the advicereceived from the Consultation.

At the request of WHO, a survey had been carried out to assess theextent of use of the established reference materials in this area. Al-though demand does not necessarily reflect therapeutic importance,the materials appear to have a significant role and are used in both thedeveloped and developing countries. However, the procedures forobtaining this information are not ideal. Nevertheless, the informa-tion on use of these standards is useful for aiding decisions aboutcapacity building.

Human interferon beta

The Committee was informed that the current International Standardfor Interferon beta is an impure preparation derived from humanfibroblasts containing about 1% interferon, and that other cytokinesthat are present influence the results of some assays. The Committeewas also informed that an extensive collaborative study of new andexisting reference preparations for interferon beta had been per-formed by 16 laboratories in eight countries. The aims of the studywere to assess the relative activities of preparations of natural andrecombinant interferon beta in a range of bioassays; to compare theseactivities where possible with those of well-characterized in-housestandards; to assess whether the current International Standard re-mains suitable; to identify candidate replacement preparations; toobtain data about assays in current use and to prepare for a separatestudy in which a single assay design would be used. A total of eightampouled preparations of interferon beta were examined. The Com-mittee noted that it is necessary to ampoule interferon beta in thepresence of casein to prevent adsorption to the glass. One candidatepreparation, consisting of glycosylated interferon beta derived fromChinese hamster ovary cells, gave a smaller inter-laboratory variabil-ity than the current standard, with all but one of the samples exam-ined. The Committee reviewed the results of the collaborative studyand a proposal to establish a replacement International Standard forFibroblast Interferon beta (WHO/BS/03.1976). On the basis of theinformation supplied, the Committee established the preparation, inampoules coded 00/572, as the Third International Standard forInterferon beta, Human, Recombinant, Glycosylated, and assigned apotency to it of 40000 International Units per ampoule. This standardreplaces the Second International Standard for Interferon, beta,Fibroblast. Since stocks of the current standard remained, the

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Committee formally disestablished the Second International Stan-dard for Interferon beta, Fibroblast, Human, code number Gb23-902-531. The material derived from Chinese hamster ovary cells cell islikely to continue to be available and is more suitable for calibrationof future therapeutic products than the fibroblast material. However,it is not suitable for assay of the Ser-17 interferon beta analogue andthe First International Standard for Interferon-beta Ser 17 mutein,code number Gxb02-901-535, will be retained.

Tumour necrosis factor alpha, human

The Committee was informed that stocks of the current InternationalStandard for Tumour Necrosis Factor Alpha, Human are almost ex-hausted and the remaining stock is required to ensure traceability inthe future. The Committee was also informed that three other candi-date preparations had been included in the collaborative study lead-ing to establishment of the current standard in 1991. Stability studiesat that time had indicated high thermal stability of the preparationsand this has been confirmed by additional studies performed during2002 and 2003. The Committee noted a proposal to establish areplacement International Standard for Tumour Necrosis Factor,Alpha, Human (WHO/BS/03.1981), based on the results of the earliercollaborative study performed by 20 laboratories in nine countriestogether with results obtained in recent bridging studies in one labo-ratory using three bioassays. The proposed standard consists of thefull-length 157-amino-acid protein, but is derived from different cellsto those used for production of the full-length 157-amino-acid proteinin the First International Standard. The other two candidate prepara-tions have different amino acid sequences. On the basis of the infor-mation supplied, the Committee established the preparation, inampoules coded 88/786, as the Second International Standard forTumour Necrosis Factor, Alpha, Human and assigned a potency to itof 46500 International Units per ampoule. Since the standard is likelyto be used in assays to measure anti-tumour necrosis factor activity,the memorandum dispatched with the standard preparation shoulddraw attention to possible discontinuities in results when compared tothose obtained with the International Standard. The recommendedInternational Nonproprietary Name (INN) for the material inthe standard should be included in the memorandum distributedwith it.

Luteinizing hormone, recombinant

The Committee was reminded that luteinizing hormone (LH) is a twosubunit glycoprotein gonadotrophin obtained from pituitary glands,

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from urine or by recombinant methods. LH is used for diagnosis andfor therapy. Because there are biological, immunological and physico-chemical differences between pituitary and urinary hormones, severalWHO international standards have been established. The Committeenoted that recombinant LH is now available as a therapeutic productand noted a proposal to establish a standard for it for use in bioassaysfor therapeutic products (WHO/BS/03.1983). When the candidatepreparation is calibrated against urinary LH and pituitary LH differ-ent values are obtained. The recombinant material most closely re-sembles pituitary LH, but it is proposed that the value determinedrelative to the urinary LH standard be adopted to ensure continuity ofunitage for therapeutic products. On the basis of the informationsupplied, the Committee established the preparation, in ampoulescoded 96/602, as the First International Standard for Luteinizing Hor-mone, Recombinant and assigned an activity to it of 189 InternationalUnits per ampoule. The Committee recommended that the recom-mended INN for the material in the standard, lutropin alfa, is in-cluded in the memorandum distributed with it.

Thyroid-stimulating hormone for immunoassay

The Committee was reminded that immunoassays for thyroid-stimulating hormone are widely employed in the diagnosis and man-agement of thyroid dysfunction. The Committee was informed thatstocks of the current Second International Reference Preparation forThyroid-Stimulating Hormone are depleted. The Committee was alsoinformed that it had been demonstrated that preparations of recom-binant thyroid-stimulating hormone are unsuitable for calibration ofdiagnostic assays. The Committee noted a proposal to establish areplacement standard for thyroid-stimulating hormone based on acollaborative study carried out by nine laboratories in six countries(WHO/BS/03.1975). The candidate preparation was obtained fromthe same bulk material of pituitary origin as the current preparationand had been included in the collaborative study when the currentpreparation was established. The present study was designed to com-pare the candidate preparation with the current preparation. Thestudy confirmed that its activity and stability have remained un-changed. On the basis of the information supplied, the Committeeestablished the preparation, in ampoules coded 81/565, as the ThirdInternational Standard for Thyroid-stimulating Hormone, Human,for Immunoassay and assigned an activity to it of 11.5 milli-International Units per ampoule.

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Interferon neutralizing antibody tests

The Committee was informed that interferons alpha and beta caninduce antibodies when used therapeutically. Such antibodies maymake the patients resistant to further treatment with interferons.However, many different methods are used to determine levels ofanti-interferon antibodies and the data obtained in one laboratorycannot necessarily be made use of in another. A standard methodfor the calculation and reporting of the results of interferon neutraliz-ing antibody tests has been proposed (WHO/BS/03.1980). However,the September 2003 WHO Consultation on cytokines (see p.22) con-sidered that, although the approach proposed was promising, moredata should be obtained and the investigators should be encouragedto use WHO reference materials to determine the sensitivity ofthe assay procedures employed. The Committee endorsed theserecommendations.

Diagnostic reagents

WHO consultation on international standards for testingdiagnostic kits used in detection of hepatitis B surface antigenand anti-hepatitis C virus antibodies

The Committee was informed of the meeting of a WHO WorkingGroup on International Reference Preparations for testing diagnostickits used in the detection of hepatitis B surface antigen (HbsAg) andanti-hepatitis C virus (HCV) antibodies held in Geneva in October2003.

The Working Group reviewed in detail a collaborative study toestablish a replacement International Standard for HBsAg (see p.27).Following discussion, the participants agreed that the candidatereplacement standard for HbsAg should be assigned a unitage inInternational Units and that the use of “nanograms” should bediscontinued. The Working Group also agreed that it had been shownthat the candidate materials were commutable between assays. TheWHO International Standard for HBsAg is intended for the calibra-tion of secondary HBsAg reference materials by manufacturers andnational regulatory authorities. The Working Group also recom-mended the establishment of a reference panel for use in the assess-ment of analytical quantitation of assay kits. The WHO ReferencePanel should aid national regulatory authorities in evaluating theanalytical sensitivity of rapid tests for HBsAg, particularly wherenegative diluent is scarce. Nevertheless it was noted that the evalua-tion of test kits for HBsAg should include performance testing of

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seroconversion panels, difficult samples, and local samples represent-ing the prevalent hepatitis B virus genotypes and variants in thattarget region.

The Group also considered that there is a role for a panel of monospe-cific anti-HCV sera to define analytical specificities of test kits foranti-HCV antibodies, and that a feasibility study of candidate materi-als should be performed. In the past the WHO Working Group onReference Standards for Hepatitis and HIV Diagnostic Kits haddesigned, produced and tested a candidate anti-HCV genotype 1breference. However, when the Working Group proposed to theExpert Committee on Biological Standardization that this prepara-tion be used as a WHO Reference Material, the suggestion was notfavourably received by the Committee because the characterizationof the serum pool was insufficient to permit determination of theanalytical sensitivity of kits that detect several antigens. In 2001, theWorking Group agreed to prepare a reference material for each offour antibodies deemed appropriate for detection by the commercialkits most frequently manufactured: anti-core, anti-NS3, anti-NS4 andanti-NS5. In early 2003, a potential source of such materials had beenidentified. The limitations of a sensitivity panel of monospecific anti-bodies for evaluating diagnostic kits worldwide were discussed. It wasagreed that to define kit performance fully, regulatory authorities andmanufacturers should use collections of positive specimens from localpopulations and seroconversion panels when available. However, theparticipants agreed that a monospecific international reference panelwould have unique value in providing a benchmark to evaluate thequality of assay kits worldwide. The value of such a panel should betested with different genotypes. It was agreed that a protocol shouldbe drafted for the performance of a feasibility study involving thecandidate materials identified. The protocol should include thesamples to be tested, the test kits with which they are to be evaluatedand the responsibility of each collaborating centre in the study.

The Committee endorsed the conclusions from the Working Group.

Hepatitis B surface antigen

The Committee was reminded of the need to replace the current FirstInternational Standard for Hepatitis B Surface Antigen. A collabora-tive study had been performed to assess the suitability of a candidatereplacement preparation and to calibrate it (WHO/BS/03.1987). Aplasma pool containing HBsAg subtype adw2, genotype A, was inac-tivated, purified and diluted. Also, a series of four fourfold dilutionsof the purified material was prepared. The first preparation was

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proposed as a candidate to replace the First WHO InternationalStandard for HBsAg, and the panel of dilutions was proposed as aWHO Reference Panel for HBsAg. The aim of these reference mate-rials is to aid regulatory authorities and manufacturers of HBsAg testkits in measuring the analytical sensitivity of kits by providing astandard with an internationally accepted unitage.

WHO collaborative studies were conducted to characterize and assessthe candidate International Standard and the proposed ReferencePanel. In order to assign an appropriate unitage, the study analysedthe candidate International Standard preparation against the FirstInternational Standard and four other widely recognized HBsAgreference standards: the primary and current Paul Ehrlich Institutestandards, a French standard and a standard used by an in vitrodiagnostics manufacturer (Abbott Laboratories). The assigned valuesof the different HBsAg reference preparations were found to differconsiderably: 1 IU is equivalent to 0.58 Paul Ehrlich Institute (PEI)units (primary) or 0.43 PEI units (current) or 1.9 French “ng” or 5.6Abbott “ng”. However, it is noteworthy that in 1985, the relationshipbetween IS units and the primary PEI units was almost the same asthat found in the current study: 1 IU = 0.55 PEI unit. These andrelated biochemical data indicate that there has been no drift in theIU over 18 years.

The overall mean IU of the candidate International Standard was33 IU/vial. Because the panel was manufactured in a series of fourfolddilutions of the candidate preparation, panel components A, B, C andD, were estimated to contain 8.25, 2.06, 0.52 and 0.13IU/vial, respec-tively. These values were confirmed experimentally for all but panelcomponent A, the values for which were out of the analytical rangefor the kits used in the quantitative phase of the study and were thustechnically invalid. In a second phase of the study, 10 internationallaboratories tested the candidate International Standard and refer-ence panel using 20 other immunoassays and rapid tests. The studyshowed that the prediluted panel provides a convenient resource foruse by regulatory authorities to assess sensitivity, especially of rapidtests.

On the basis of the results obtained, the Committee established thecandidate preparation, in vials coded 00/588, as the Second Interna-tional Standard for Hepatitis B Surface Antigen with an assignedvalue of 33 IU per vial. The standard contains antigen subtype adw2,genotype A. The Committee also established panel components A toD, in vials coded 01/400, 01/402, 01/404 and 01/406, which are 1 in 4, 1in 16, 1 in 64 and 1 in 256 dilutions of the International Standard,

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respectively, and panel component E, in vials coded 00/616, whichconsists of human re-calcified plasma, as a reference panel for HbsAgfor use by national regulatory authorities in the assessment of thesensitivity of assay kits for the detection of the surface antigen. TheCommittee reviewed the memoranda to be supplied with the refer-ence materials, and, after making some changes, approved them.

Lipoprotein (a)

The Committee was informed that the presence of high levels oflipoprotein (a) is a genetically determined marker of predisposition tocoronary artery disease. Accurate measurement of lipoprotein (a) isimportant for diagnosis and a Working Party, established by theInternational Federation of Clinical Chemistry and Laboratory Medi-cine, had performed a series of studies to calibrate the lipoprotein (a)content of pooled human serum in terms of purified preparations oflipoprotein (a). The Committee noted a proposal to establish a refer-ence material for lipoprotein (a) (WHO/BS/03.1979), based on acollaborative study performed by 18 laboratories in 10 countriesemploying two different ELISA methods. The Committee was in-formed that when the proposal had been presented at its previous(fifty-third) meeting, a request had been made for additional informa-tion. This information had now been included in the report. Thestability of the preparation had been assessed in various real-time andaccelerated studies over 5 years and appeared to be adequate. Moni-toring of stability continues. On the basis of the results obtained, theCommittee established the preparation, in vials coded SRM 2B, as theFirst WHO Reference Reagent for Lipoprotein (a) for Immunoassayand assigned a value of 0.107 nanomoles per vial to it.

Miscellaneous

Standardization of human papillomavirus vaccine

The Committee was reminded that human papillomavirus (HPV) isresponsible for a variety of diseases and is a major cause of cancer inmany women. Four strains of virus have been identified as beingassociated with cervical cancer. The Committee was also informedthat candidate vaccines consisting of papilloma-like virus particleshad been developed and studied in clinical trials and appear to besafe, immunogenic and well-tolerated and to offer complete protec-tion against HPV infections. Reference reagents are required toevaluate antibody responses to the vaccines and to monitor incidenceof disease. Collaborative studies have been performed to harmonize

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diagnostic procedures for HPV DNA type-specific detection and inserological type-specific assays and evaluate the use of reagents forthese. The outcome of the studies was reviewed at a WHO Workshopheld in Geneva in September 2003 as a result of which it was proposedthat international standards and reagents be developed for type-specific assays, beginning with DNA standards for types 16 and 18. Itwas also proposed that monotypic reference antisera to HPV types 16and 18 be submitted for adoption as international standards for sero-logical assays. These assays are usually specific for one type, but varyin sensitivity. The Committee agreed that the work proposed onreference materials should proceed and recommended that neutraliz-ing assays should be included in the antisera studies.

Standardization of human immunodeficiency virus neutralizingantibody assays

The Committee was informed that measurement of anti-HIV-1 neu-tralizing antibodies is important in HIV vaccine research and in clini-cal trials. To facilitate progress in this field, a WHO–Joint UnitedNations Programme on HIV/AIDS (UNAIDS) meeting was held inMilan, Italy, in August 2003. This revealed that a wide range of assayswere being used. It was agreed that standardization through provisionof reference materials and development of assay protocols is re-quired. It was also agreed that a collaborative study should be per-formed to evaluate candidate neutralization standards using differentassays against a panel of viral subtypes, obtained using plasma and/sor sera from different geographical areas, as well as monoclonalantibodies evaluated as potential standards. Detailed proposals willbe prepared when details of the availability of material are known.The Committee agreed with the course of action proposed.

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 1WHO guidelines on nonclinicalevaluation of vaccines

This document provides guidance to national regulatory authorities(NRAs) and vaccine manufacturers on the nonclinical evaluation of vac-cines by outlining the international regulatory expectations in this area. Itshould be read in conjunction with the Guidelines on clinical evaluation ofvaccines: regulatory expectations (1), in order to complete the under-standing of the whole process of vaccine evaluation. Vaccines area diverse class of biological products and their nonclinical testingprogrammes will depend on product-specific features and clinical indica-tions. The following text has therefore been written in the form of guide-lines rather than recommendations. Guidelines allow greater flexibilitythan recommendatisons with respect to specific issues related to particu-lar vaccines.

Introduction 32

1 General remarks 321.1 Scope 341.2 Glossary 34

2 Characterization of candidate vaccines 37

3 Immunogenicity and other pharmacodynamic studies 43

4 Toxicity assessment 444.1 Basic toxicity assessment 454.2 Additional toxicity assessments 48

5 Special considerations 515.1 Adjuvants 515.2 Additives (excipients and preservatives) 525.3 Vaccine formulation and delivery device 525.4 Alternative routes of administration 53

6 Specific considerations for particular types of vaccines 54

Authors 56

References 58

AppendixList of tissues to be collected in a repeated dose toxicity study 62

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Introduction

Recent progress in biotechnology and basic immunology has led tothe development of a broad range of novel vaccines raising excitingpossibilities for the prevention of infectious diseases (2, 3). Improve-ments to already licensed vaccines are also being considered; suchimprovements will lead to new products as well as to the introductionof new adjuvants. However, the complexity and novelty of theseproducts presents scientific and regulatory challenges because criteriafor their safety, potency and quality assessment may not exist. Prod-uct diversity and new approaches, technologies and methodologiesdevelop over time; therefore, judgement based on the best scienceavailable should always form the basis for deciding on the type andextent of nonclinical evaluation for these products.

Although nonclinical evaluation plays an essential part in theoverall development of vaccine candidates, there is at present limitedguidance regarding nonclinical evaluation programmes for theseproducts. In this guidance document, the general principles ofnonclinical evaluation of vaccines are discussed, with particular atten-tion being given to the regulatory expectations for new and novelvaccines.

Preclinical testing is a prerequisite to moving a candidate vaccinefrom the laboratory to the clinic and includes all aspects of testing,product characterization, proof of concept/immunogenicity studiesand safety testing in animals conducted prior to clinical testing of theproduct in humans. Nonclinical evaluation, within the context ofthis document, refers to all in vivo and in vitro testing performedbefore and during the clinical development of vaccines. For example,nonclinical evaluation may be necessary when changes in the manu-facturing process or product formulations are made or to furtherstudy potential safety concerns that may have arisen from phase Iand II trials or that have been described in the literature for similarproducts.

1 General remarks

Nonclinical studies are aimed at defining the in vitro and in vivocharacteristics of candidate vaccines including those relating to safetyand immunogenicity. Nonclinical studies in animals are valuable toolsfor identifying possible risks to the vaccinees and helping to planprotocols for subsequent clinical studies in human subjects. However,in all cases, when safety testing in animals is performed, there shouldbe a clear rationale for doing so and the study should be performed in

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compliance with the national and international laws for the protectionof laboratory animals (4), biosafety requirements (5) and with goodlaboratory practice (GLP) (6). However, there may be situationswhere full compliance with GLP is not possible. If the study, or partof the study, was not conducted in compliance with GLP, areas ofnoncompliance should be defined and a statement of the reason fornoncompliance should be drawn up.

Potential safety concerns for a vaccine product include those due toinherent toxicities of the product, toxicities of impurities and con-taminants, and toxicities that result from interactions between thevaccine components present in the vaccine formulation. In addition,the immune response induced by the vaccine may lead to toxicside-effects.

Despite efforts to maximize the predictive value of nonclinical toxic-ity studies there is always the possibility that not all risks are identi-fied. The limitations of animal testing in reflecting clinical safety andefficacy in humans should be recognized as pathogenesis and immuneresponses are frequently species-specific. Moreover, potential safetyconcerns identified during animal testing may not necessarily indicatea problem in humans. However, any signal observed in nonclinicaltoxicity studies should be carefully addressed in human clinical trialsand may require additional nonclinical testing. It should be notedthat the absence of detectable toxicity in animal studies does notnecessarily mean a vaccine will be safe in humans. Potential safetyconcerns related to specific types of vaccine candidate are consideredin section 6.

The development and subsequent validation of in vitro tests for use asalternatives to nonclinical evaluation of vaccine candidates in animalsis encouraged as it may lead to the improvement of nonclinical testingas well as to a reduction of animal usage.

The need for and extent of nonclinical testing will depend on theproduct under consideration. For example, for a product for whichthere is no prior nonclinical and clinical experience, nonclinical test-ing would be expected to be more extensive than for those vaccinespreviously licensed and used in humans. In some cases, it may not benecessary to perform preclinical safety studies prior to the initiationof phase 1 clinical trials. For example, in the case of transfer oftechnology, where access to the database of the originally developedvaccine is available, data from nonclinical bridging studies (e.g. physi-cochemical characterization and abbreviated in vivo studies) may bean acceptable basis for further development of the product.

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Early communication between the vaccine manufacturer and the re-sponsible national regulatory authority to agree on the requirementsfor and type of nonclinical testing is recommended.

1.1 Scope

For the purposes of this document, vaccines are considered to be aheterogeneous class of medicinal products containing immunogenicsubstances capable of inducing specific, active and protective hostimmunity against infectious disease.

Although most vaccines are being developed for pre- and post-exposure prophylaxis, in some cases, they may be indicated for thera-peutic use against infectious diseases, e.g. human immunodeficiencyvirus (HIV), and human papillomavirus (HPV). Both prophylacticand therapeutic vaccines for infectious disease indications are consid-ered in this document.

Vaccines for human use include one or more of the following: mi-croorganisms inactivated by chemical and/or physical means thatretain appropriate immunogenic properties; living microorganismsthat have been selected for their attenuation whilst retainingimmunogenic properties; antigens extracted from microorganisms,secreted by them or produced by recombinant DNA technology;chimeric microorganisms; antigens produced in vivo in the vaccinatedhost following administration of a live vector or nucleic acid or anti-gens produced by chemical synthesis in vitro. The antigens may be intheir native state, truncated or modified following introduction ofmutations, detoxified by chemical or physical means and/or aggre-gated, polymerized or conjugated to a carrier to increase immunoge-nicity. Antigens may be presented plain or in conjunction with anadjuvant, or in combination with other antigens, additives and otherexcipients.

Therapeutic vaccines for non-infectious diseases (e.g. certain cancervaccines) and monoclonal antibodies used as immunogens (e.g. anti-idiotypic antibodies) are not considered here.

1.2 Glossary

The definitions given below apply to the terms used in these guide-lines. They may have different meanings in other contexts.

AdjuvantsSubstances that are intended to enhance relevant immune responseand subsequent clinical efficacy of the vaccine.

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Booster vaccinationVaccination given at a certain time interval after primary vaccinationto enhance immune responses and induce long-term protection.

Combination vaccineA vaccine that consists of two or more antigens, either combined bythe manufacturer or mixed immediately before administration andintended to protect against either more than one disease, or againstone disease caused by different strains or serotypes of the sameorganism.

Genetically modified organism (GMO)An organism or a microorganism in which the genetic material hasbeen altered in a way that does not occur naturally by mating and/ornatural recombination. This definition covers microorganisms includ-ing viruses, viroids and cell cultures including those from animals,but does not cover naked recombinant DNA or naked recombinantplasmids.

Good clinical practice (GCP)A standard for clinical studies that encompasses their design, conduct,monitoring, termination, audit, analyses, reporting and documenta-tion and which ensures that the studies are scientifically and ethicallysound and that the clinical properties (diagnostic, therapeutic or pro-phylactic) of the pharmaceutical product under investigation areproperly documented.

Good laboratory practice (GLP)A quality system concerned with the organizational process and theconditions under which nonclinical health and environmental safetystudies are planned, performed, monitored, recorded, archived andreported. GLP principles may be considered as a set of criteria to besatisfied as a basis for ensuring the quality, reliability and integrity ofstudies, the reporting of verifiable conclusions and the traceability ofdata.

Good manufacturing practice (GMP)A part of the pharmaceutical quality assurance which ensures thatproducts are consistently produced and controlled according to thequality standards appropriate to their intended use and as required bythe marketing authorization. In these guidelines, GMP refers to thecurrent GMP guidelines published by WHO.

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ImmunogenicityCapacity of a vaccine to induce antibody-mediated and/or cell-mediated immunity and/or immunological memory.

Nonclinical evaluation of vaccinesAll in vivo and in vitro testing performed before and during clinicaldevelopment of vaccines. The potential toxicity of a vaccine should beassessed not only prior to initiation of human trials, but throughoutclinical development.

PlasmidDouble-stranded circular DNA molecules capable of replicating inbacterial cells.

PotencyThe measure of biological activity, using a suitable quantitative bio-logical assay, based on the attribute of the product that is linked to therelevant biological properties.

Preclinical evaluation of vaccineAll in vivo and in vitro testing carried out prior to the first testing ofvaccines in humans. This is a prerequisite to the initiation of clinicaltrials and includes product characterization, proof of concept/immu-nogenicity studies and animal safety testing.

Preclinical toxicity studyA study designed with the primary purpose of demonstrating thesafety and tolerability of a candidate vaccine product. The design ofthe preclinical toxicity study should meet the criteria outlined in thesection on study design to be considered supportive of the intendedclinical trial.

Primary vaccinationFirst vaccination or series of vaccinations given within a predefinedperiod, with an interval of less than 6 months between doses, toinduce clinical protection.

Product characterizationA full battery of physical, chemical and biological tests conducted fora particular product. These tests include, but are not limited to, in-process control testing, testing for adventitious agents, testing processadditives and process intermediates, and lot release.

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Protocol or study planA document that states the background, rationale and objectives ofthe nonclinical studies and describes its design, methodology andorganization, including statistical considerations, and the conditionsunder which it is to be performed and managed.

Relevant animal modelAn animal that develops an immune response similar to the expectedhuman response after vaccination. It is acknowledged that species-specific differences in immune responses are likely. Ideally, the ani-mal species chosen should be sensitive to the pathogenic organism ortoxin under consideration.

Route of administrationThe means by which the candidate vaccine product is introduced tothe host. Possible routes of administration include the intravenous,intramuscular, subcutaneous, transcutaneous, intradermal, trans-dermal, oral, intranasal, intranodal, intravaginal and intrarectalroutes.

SeroconversionPredefined increase in antibody concentration, considered to corre-late with the transition from seronegative to seropositive, providinginformation on the immunogenicity of a vaccine. If there are pre-existing antibodies, seroconversion is defined as a transition from apredefined low level to a significantly higher defined level, such as afourfold increase in geometric mean antibody concentration.

ValidationThe action of proving, in accordance with the principles of goodmanufacturing practice, that any procedure, process, equipment(including the computer software or hardware used), material, activ-ity or system actually leads to the expected results.

2 Characterization of candidate vaccines2.1 Vaccine production

The biological nature of the starting materials, the manufacturingprocess and the test methods needed to characterize batches of theproduct are important elements to be considered in the design and theinterpretation of nonclinical testing of vaccines. Many vaccines areproduced using prokaryotic or eukaryotic microorganisms and subtlechanges in these organisms may radically affect the vaccine product.Therefore, the establishment of a seed-lot system is essential forvaccine production. Moreover, the quality, safety and potency of

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these products are usually sensitive to changes in manufacturingconditions. The quality and safety of vaccine preparations cannot beassured solely by testing of the end-product, but depends on the strictcontrol of the manufacturing process following the principles of goodmanufacturing practice (GMP) (7). This includes demonstration ofthe purity and quality of the starting material (raw materials andseeds), in-process control testing, testing for process additives andprocess intermediates and the development and establishment of lotrelease tests. Moreover, as the relationship between physical andchemical characteristics, and the immunogenicity and efficacy ofthese products is frequently not completely understood, biologicalcharacterization through the use of biological assays should alwayscomplement the physical and chemical product characterization. Thedevelopment of appropriate laboratory methods to characterize avaccine formulation with respect to its components, as well as itssafety and potency, is a prerequisite to the clinical use of any new ornovel vaccines against bacteria, viruses or parasites.

Consistency of production is essential, and the demonstration that theproduct does not differ from vaccine lots that have been shown to besafe and adequately immunogenic and protective in clinical studiesis a crucial component of vaccine evaluation, licensing and batchrelease. For this reason, manufacturers should make every effort tocharacterize these clinical lots and if possible to keep some of theselots for future reference.

Where no appropriate animal model exists for testing potency orwhere direct serological or immunological correlates of clinical pro-tection are not available, the challenge is to ensure that each produc-tion batch has the same protective efficacy as those batches shown tobe protective in clinical trials. In such cases, emphasis is increasinglybeing placed on assuring the consistency of production using modernphysical, chemical and immunological methods that enable character-ization of some products to a degree of precision not previouslypossible.

The vaccine lots used in preclinical studies should be adequatelyrepresentative of the formulation intended for use in the clinicalinvestigation and, ideally, preclinical testing should be done on thesame lot as that proposed for the clinical trials. If this is not feasible,then the lots studied should be comparable with respect to physico-chemical data, stability and formulation.

At a minimum, candidate vaccines for clinical trials should be pre-pared under conditions of good manufacturing practice (GMP) for

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clinical trial material (8). However full GMP will be required at thelater stages of clinical development (7, 9).

Any change proposed to the manufacturing process during vaccinedevelopment should be considered carefully to evaluate its impact onthe quality, safety and efficacy of the vaccine and the possible need foradditional nonclinical and clinical investigations.

Subsequent changes in production methods or scale-up followingproduct licensure will necessitate further product characterization todemonstrate comparability with the original lot(s) used to demon-strate safety and efficacy of the product. The extent of comparabilitytesting needed depends on the nature of the changes implemented(10). These changes should be documented and the national regula-tory authority consulted. Regulatory authorities should clearly defineand implement in their regulations what changes require only anotification and which changes require formal approval before imple-mentation (11).

The procedures used in the characterization and control of existinglicensed traditional vaccines are not likely to be applicable to newerproducts developed using state-of-the-art technology to protectagainst the same infection. For example, specific guidelines have beendeveloped for the production and control of acellular pertussis vac-cines that differ from those applied to whole cell pertussis vaccine(12). Likewise, the tests applied to the characterization and control oftraditional inactivated cholera vaccine for parenteral use are not nec-essarily applicable to the new inactivated whole-cell cholera vaccineintended for oral administration, and an appropriate potency test forthe oral vaccine needs to be developed.

2.2 Potency

Potency tests measure the biological activity of a vaccine but do notnecessarily reflect the mechanism of protection in humans. Potencymeasurement is often used to verify the consistency of the manufac-turing process. The initial concept of potency testing for vaccines wasto quantify the biological activity of the vaccine in comparison with areference preparation of known bioactivity, where the antigeniccomponent(s) were not well-defined.

Classical challenge studies in animals immunized with the vaccineunder consideration have been developed into routine potency assays(e.g. for diphtheria and tetanus toxoids). In the case of the whole-cellpertussis potency assay, which consists of intracerebral challenge ofimmunized and nonimmunized animals, a correlation was establishedwith clinical protection in humans (11). Where no suitable animal

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challenge model exists, potency is often based on measurement ofimmune responses, usually serological (e.g. influenza and hepatitis Bvaccines).

More recently, recombinant DNA methodology and modern physico-chemical techniques have resulted in the manufacture of highly puri-fied products that can be better characterized than the classicbiologicals. However, the ability to measure the “relevant” biologicalactivity for such products may still be lacking. For these products,characterization using physicochemical parameters, such as amountof antigen, size of the antigen, protein content and others can be usedas a measure of consistency, but not necessarily of the potency of avaccine.

For live attenuated vaccines, the approach to potency measurement isgenerally different. The potency of live viral vaccines is usually basedon titration of the minimum infective dose in cell culture or chickenembryos, which may be considered as a surrogate marker of potency,but not as a measure of potency itself. A similar approach is takento the potency measurement of live attenuated bacterial vaccines,bacille Calmette–Guérin (BCG), and typhoid vaccine (live Ty21Aoral), where the number of live organisms present is the measure ofpotency.

For vaccines that express inserts encoding heterologous vaccine anti-gens (vaccines based on viral or bacterial vectors), it is not sufficientto determine the “biological activity” of the entire construct by mea-suring colony forming units (CFU) or infectious titre. For thesevaccines, the use of other methods such as the quantitation of theexpression of the insert, or the evaluation of the effective dose (ED50)of the vectored vaccine should be considered.

2.3 Stability

The evaluation of vaccine stability is complex, as they are very suscep-tible to inactivation by environmental factors. Potency, as defined inthe glossary, should be measured as a part of the stability testing,except in those cases where potency testing based on biological activ-ity is not possible. Physical and chemical product characterizationshould be included in the stability evaluation. For a product enteringhuman clinical trials, sufficient data should be collected to supportthe stability of the product for the duration of the preclinical andclinical trial. In certain cases, accelerated stability data may be used tosupport preliminary data obtained at the normal storage temperature.Stability data to support licensure should be obtained under theproposed storage conditions and should be based on long-term,

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real-time stability studies. Finally, the stability of standards andreference materials also needs to be considered to ensure that theprocedures used to measure relevant parameters are reliablystandardized.

2.4 International and national guidelines

The World Health Organization (WHO), through considerable inter-national consultation, develops Recommendations and Guidelineson the production and control of vaccines and other importantbiologicals (13), and these form the basis for assuring the acceptabilityof products globally. These documents specify the need for appropri-ate starting materials, including seed lot system and cell banks; strictadherence to established protocols; tests for purity, potency, andsafety at specific steps during production; and the keeping of properrecords. Guidelines allow greater flexibility than Recommendationswith respect to specific issues related to particular vaccines.

WHO also provides Guidelines on manufacturing establishments in-volved in vaccine production. Recommendations can be found in theWHO document on good manufacturing practice for biologicals (7).Particular attention should be given to developing documented stan-dard operating procedures for both production processes and testingprocedures. These should be introduced as early as possible duringthe development of a vaccine and be well established by the timephase III clinical studies are undertaken and an application for mar-keting authorization is filed. The basic principles for the productionand control of vaccines are published in the WHO Technical ReportSeries (7, 14–18). Specific WHO guidelines and recommendations forparticular vaccines are also available and should be consulted whereappropriate.

WHO Recommendations and Guidelines are intended to be scientificand advisory in nature and to provide guidance for national regula-tory authorities and for vaccine manufacturers. These documents maybe adopted by national health authorities as definitive national regu-lations or used as the basis of such regulations. They are also used asthe basis for deciding the acceptability of vaccines for purchase byUnited Nations agencies such as the United Nations Children’s Fund(UNICEF) for use in global immunization programmes. Regulatoryrequirements for vaccines and other biologicals are also produced byother bodies, such as the European Agency for the Evaluation ofMedicinal Products (EMEA) and the US Center for Biologics Evalu-ation and Research (CBER) (19); these documents can be found onthe appropriate web sites (www.emea.eu.int and www.fda.gov/cber).In addition, pharmacopoeial requirements, such as those of the

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European Pharmacopoeia, are also established for vaccines and areavailable at www.pheur.org.

For newly developed products, specific WHO, national or pharmaco-poeial requirements may not be available and a national regulatoryauthority will need to agree on specifications with the manufactureron a case-by-case basis during the evaluation of products for clinicaltrials and for licensing. For some of these novel products generalguidance on production and control from WHO can be found inrelevant documents, such as those describing DNA and peptide vac-cines (14, 16), as well as recommendations on animal cell substratesused for production of biologicals (14).

In addition, information on how to assure the quality of biologicals ingeneral and on procedures for approving manufacture and for settingup a national control laboratory, can be found in the relevant WHOguidelines (17, 18). For a vaccine intended to be marketed worldwide,the development of which also involves much international collabora-tion, it will be essential to ensure consistency of a regulatory approachfor novel products such as vaccines for HIV prevention (19).

2.5 Batch release and independent laboratory evaluation

The potential variability of methods for the production of biologicalshas led to the establishment of national and international require-ments to define procedures for assuring the quality of vaccines andfor assessing consistency both among manufacturers and over longperiods of time. Licensed vaccines are subject to independent batchrelease (review, testing and authorizing release of a batch of vaccineindependent of the manufacturer) by a national regulatory authorityor national control laboratory, before release on to the market. Inde-pendent evaluation entails at least an evaluation of a manufacturer’sbatch release data (protocol review), but in many instances it alsoincludes independent laboratory testing in addition to that carried outby the manufacturer.

Batch or lot release tests are those tests chosen during full productcharacterization to demonstrate the purity, safety and potency ofthe product. Lot release testing provides one measure of assurancethat a lot can be manufactured consistently. Validation and establish-ment of lot release tests and specifications is a process that continuesthroughout product development and should be finalized prior tolicensure.

In some countries, samples of vaccine for clinical trials are required bythe national regulatory authority, as a part of the approval process forclinical trials. Vaccine developers are encouraged to consult the

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appropriate regulatory agency early on during the development of avaccine.

2.6 Standards and reference materials

Standards and reference materials play a vital part in the licensing andquality control process, their role ranging from use in specific antigenrecognition tests to assays of vaccine toxicity, immunogenicity andpotency. The standardization of the methods used to evaluate vac-cines, as well as those used to evaluate immune responses to vaccineantigens, is also vital so that results may be compared directly be-tween laboratories both within and between countries, and betweenclinical trials.

WHO International Biological Standards and Reference Reagentsare the primary standards in use worldwide. In addition, nationalregulatory authorities and manufacturers may establish secondary(regional, national), working standards for the purpose of testingvaccine quality on a lot-to-lot basis. Such standards should be cali-brated against International Standards, when they exist. There isconcern that different secondary standards may result in “drifting”from the International Standard. Production of secondary standardson a large scale (e.g. on a regional basis) reduces the number ofsecondary standards in use, and should improve accuracy of testingvaccine quality. For example, the European Department for theQuality of Medicines of the Council of Europe, has been active inestablishing working standards for vaccines that are calibrated againstthe WHO International Standards, where appropriate. The completelist of WHO International Standards and Reference Reagents can befound on the WHO web site at: www.who.int/ biologicals.

3 Immunogenicity and other pharmacodynamic studies

A pharmacodynamic study for a vaccine product is generally con-ducted to evaluate the immunogenicity. However, a pharmacody-namic study may also extend to include the pharmacology of anadjuvant.

Immunization studies in animal models should be conducted becausethey may provide valuable “proof of concept” information to supporta clinical development plan. In addition, immunogenicity data de-rived from appropriate animal models are useful in establishing theimmunological characteristics of the product and may guide selectionof the doses, schedules and routes of administration to be evaluatedin clinical trials. Nonclinical immunogenicity studies should assessthe relevant immune response, e.g. humoral and/or cell-mediated

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immune response, induced in the vaccinated animals. Depending onthe immune response induced, such studies may include an evaluationof seroconversion rates, geometric mean antibody titres, or cell-mediated immunity in vaccinated animals. Nonclinical studies should,where possible, be designed to assess relevant immune responses,including functional immune response (e.g. neutralizing antibodies,opsonophagocytic activity, etc.) leading to protection. These studiesmay also be designed to address interference between antigens and/orlive viruses. If a vaccine consists of more than one defined antigen(e.g. acellular pertussis vaccine consisting of 3–5 protein products) theresponse to each antigen should be evaluated. Where appropriate,challenge/protection studies with the corresponding infectious agentmay be conducted to confirm the relevance of the animal models.A primary concern in interpreting the data obtained from such studiesshould be to determine how closely the animal model resembles thedisease and immune response in humans. It should be recognized thatanimal models frequently fail to predict immunogenicity and efficacyin humans.

4 Toxicity assessment

The nonclinical safety assessment of vaccines needs to be viewed inthe context of the evolving field of vaccine development. Thus, judge-ment based on the best science available should always form the basisfor any decisions regarding the need for nonclinical safety studies,types of study and study designs. Similarly, scientific judgementshould be applied to the interpretation of data from preclinical stud-ies, regarding the risk–benefit ratio, animal model, dosing etc. Forexample, the observation of hypersensitivity reactions in an animalmodel may not necessarily preclude proceeding to clinical trials, butmay indicate the necessity for careful monitoring of a particular clini-cal parameter.

Section 4.1 provides a general framework for designing a preclinicaltoxicity study for a vaccine. The parameters set out in this section areconsidered the minimum necessary for a safety assessment prior tothe initiation of clinical trials in humans, in situations where preclini-cal safety studies are deemed necessary. As the design of any toxicitystudy is product-specific and based on indications, modifications tothe framework outlined below may be necessary in response to par-ticular product features, availability of animal models, methodolo-gies, etc.

Section 4.2 provides additional considerations for performingspecial toxicity assessments that may be required on a case-by-casebasis.

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4.1 Basic toxicity assessment4.1.1 Study design

The preclinical toxicity study should be adequate to identify andcharacterize potential toxic effects of a vaccine to allow investigatorsto conclude that it is reasonably safe to proceed to clinical investiga-tion. The parameters to be considered in designing animal toxicologystudies are the relevant animal species and strain, dosing schedule andmethod of vaccine administration, as well as timing of evaluation ofend-points (e.g. sampling for clinical chemistry, antibody evaluationand necropsy). The route of administration should correspond tothat intended for use in the clinical trials. When the vaccine is tobe administered in human clinical trials using a particular device, thesame device should be used in the animal study, where feasible (e.g.measles aerosol vaccine in the monkey model). Potential toxic effectsof the product should be evaluated with regard to target organs, dose,route(s) of exposure, duration and frequency of exposure, and poten-tial reversibility. The toxicity assessment of the vaccine formulationcan be done either in dedicated-stand alone toxicity studies or incombination with studies of safety and activity that have toxicity end-points incorporated into the design. The study should also include anassessment of local tolerance.

4.1.2 Animal species, sex, age and size of groupsData to be recorded on the animals used for toxicity testing shouldinclude information on the source, species and animal husbandryprocedures (e.g. housing, feeding, handling and care of animals). Ingeneral, the use of outbred animals is recommended. The health ofthe animal will need to be evaluated in accordance with acceptableveterinary medical practice to ensure that animals are free of anycondition that might interfere with the study. For instance, individualhousing of laboratory animals may be required to minimize the risk ofcross-infection.

Where possible, the safety profile of a product should be character-ized in a species sensitive to the biological effects of the vaccine beingstudied. Ideally, the species chosen should be sensitive to the patho-genic organism or toxin. The animal species used should develop animmune response to the vaccine antigen. In general, one relevantanimal species is sufficient for use in toxicity studies to support initia-tion of clinical trials. However, there may be situations in which twoor more species may be necessary to characterize the product, forexample where the mechanism of protection induced by the vaccine isnot well understood (for example, intranasal influenza vaccine andintranasal measles vaccine).

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In addition, when species-specific or strain-specific differences in thepharmacodynamics of the product are observed, it may be necessaryto address the nonclinical safety of the product in more than onesafety study and in more than one animal model.

The size of the treatment group depends on the animal model chosen.The number of animals used in studies using non-human primateswould be expected to be less than that in studies that used rodents.For small animal models, e.g. rats and mice, it is recommended thatapproximately 10 males + 10 females per group be studied.

In general, the approximate age at the start of the study for rodents is6–8 weeks, and for rabbits, 3–4 months.

4.1.3 Dose, route of administration and control groupsThe toxicity study should be performed using a dose that maximizesexposure of the animal to the candidate vaccine and the immuneresponse induced, for example, peak antibody response. In general,an evaluation of the dose–response is not required as part of the basictoxicity assessment and the lethal dose does not have to be deter-mined. However, pilot dose–response studies may be conducted todetermine which dose induces the highest antibody production in theanimal model. If feasible, the highest dose (in absolute terms) to beused in the proposed clinical trial should be evaluated in the animalmodel. However, the dose is sometimes limited by the total volumethat can be administered in a single injection, and guidelines onanimal welfare should be followed. In such cases, the total volumemay be administered at more than one site using the same route ofadministration. Alternatively, a dose that exceeds the human dose ona mg/kg basis and that induces an immune response in the animalmodel may be used. In such cases, the factor between human andanimal dose should be justified.

The number of doses administered to the test animals should be equalto or more than the number of doses proposed in humans. To bettersimulate the proposed clinical usage, vaccine doses should be given atdefined time intervals rather than as daily doses; the dosing intervalused in the toxicity study may be shorter (e.g. an interval of 2–3weeks) than the proposed interval in clinical trials in humans. Thedosing interval in nonclinical trials may be based on the kinetics of theprimary and secondary antibody responses observed in the animalmodel. A single-dose study may be performed in situations in whichvaccine-induced antibodies are expected to neutralize a live viralvector, thus limiting the expression of the gene of interest (e.g. anti-adenovirus immune response), or when immune responses induced in

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animals are expected to react with species-specific proteins present inthe vaccine formulation (e.g. human recombinant cytokines used asadjuvants).

The route of administration should correspond to that intended foruse in the human clinical trials. If toxic effects are observed in safetystudies using a particular route of administration (e.g. intranasal),further toxicity studies using a different route of administration (e.g.intravenous) may be helpful in understanding the full spectrum oftoxicity of the product.

The study design should include a negative control group(s) to evalu-ate a baseline level of treatment. If appropriate, active control groups(e.g. vaccine formulation without antigen) may also be included in thestudy. The study should include an additional treatment group ofanimals to be killed and evaluated as described below at later time-points after treatment, to investigate the reversibility of any adverseeffects observed during the treatment period and to screen for pos-sible delayed adverse effects.

4.1.4 Parameters monitoredToxicity studies should address the potential of the product for caus-ing local inflammatory reactions, and possible effects on the draininglymph nodes, systemic toxicity and on the immune system. A broadspectrum of information should be obtained from the toxicity studies.Parameters to be monitored should include daily clinical observa-tions, weekly body weights and weekly food consumption. Duringthe first week of administration frequent measurements of bodyweight and food consumption are recommended, if feasible, as theseare sensitive parameters indicating “illness”. Interim analysis ofhaematology and serum chemistry should be considered approxi-mately 1–3 days following the administration of the first and last doseand at the end of the recovery period. Haematology and serum chem-istry analyses should include, at the minimum, an evaluation of rela-tive and absolute differential white blood cell counts (lymphocytes,monocytes, granulocytes, abnormal cells) and albumin/globulin ratio,enzymes and electrolytes. In some cases, it may also be useful toevaluate coagulation parameters, urine samples and serum immuno-globulin classes. Data should be collected not only during treatment,but also following the recovery phase (e.g. 2 weeks or more followingthe last dose) to determine persistence, and look at exacerbationand/or reversibility of potential adverse effects.

At study termination, final body weights (after a period of fasting)should be measured. Terminal blood samples should be collected and

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serum chemistry, haematology and immunological investigationsshould be done as described in the preceding paragraph. The immuneresponse induced by the candidate vaccine should be assessed inorder to confirm that the relevant animal model has been selected. Acomplete gross necropsy should be conducted and tissues collectedand preserved, gross lesions should be examined and organ weightsrecorded (23). Histopathological examinations of tissues shouldbe performed and special attention paid to the immune organs, i.e.lymph nodes (both local and distant from site of administration),thymus, spleen, bone marrow and Peyer’s patches or bronchus-associated lymphoid tissue, as well as organs that may be expected tobe affected as a result of the particular route of administration chosen.Histopathological examinations should always include pivotal organs(e.g. brain, kidneys, liver and reproductive organs) and the site ofvaccine administration. The choice of tissues to be examined (rangingfrom a short list limited to immune and pivotal organs to a full listas provided in the Appendix) will depend on the vaccine in ques-tion, and the knowledge and experience obtained from previousnonclinical and clinical testing of the vaccine components. For ex-ample, full tissue examination will be required in the case of novelvaccines for which no prior nonclinical and clinical data are available.Therefore, the list of tissues to be tested should be defined on a case-by-case basis, following consultation with the relevant regulatory au-thority. Data should be reported in full listing the original collectionof values, and summarized.

4.1.5 Local toleranceThe evaluation of local tolerance should be conducted either as a partof the repeated dose toxicity study or as a stand-alone study. Toler-ance should be determined at those sites that come into contact withthe vaccine antigen as a result of the method of administration, andalso at those sites inadvertently exposed (e.g. eye exposure duringadministration by aerosol) to the vaccine. More details have beenpublished elsewhere (24).

If abnormalities are observed in the basic toxicity study outlined insection 4.1., further studies may be necessary to evaluate the mecha-nism of the toxic effect.

4.2 Additional toxicity assessments4.2.1 Special immunological investigations

In certain cases, the results from evaluations of immune responsefrom nonclinical and clinical studies, or from data on natural disease,may indicate immunological aspects of toxicity, e.g. precipitation of

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immune complexes, humoral or cell-mediated immune responseagainst antigenic determinants of the host itself as a consequence ofmolecular mimicry or exacerbation of the disease (e.g. inactivatedmeasles vaccine). In such cases, additional studies to investigate themechanism of the effect observed might be necessary.

Great similarity of vaccine determinants and host molecules couldcause autoimmune reactions induced by molecular mimicry (26).Therefore, any vaccine antigen whose characteristics might mimicthose of a host antigen should be treated with caution, even though itis recognized that molecular mimicry does not necessarily predisposeto autoimmunity.

Because considerable efforts may be required in selecting and devel-oping relevant animal models to address the above issues, cautionshould be exercised and a strong rationale provided when developingvaccines for diseases associated with autoimmune pathology.

If data suggest that the pathogen against which the vaccine is directedmay cause autoimmune pathology, studies may be needed to addressthis concern on a case-by-case basis, if an appropriate animal modelexists.

It should be noted that observations of biological markers for auto-immune reactions are not necessarily linked to pathogenic conse-quences. For instance, the presence of autoimmune antibodies doesnot necessarily indicate the induction of autoimmune disease (25).

When hypersensitivity reactions induced by the antigen(s), adjuvants,excipients or preservatives are of concern, additional investigationsmay be warranted.

4.2.2 Developmental toxicity studiesDevelopmental toxicity studies are usually not necessary for vaccinesindicated for immunization during childhood. However, if the targetpopulation for the vaccine includes pregnant women and women ofchildbearing potential, developmental toxicity studies should be con-sidered, unless a scientific and clinically sound argument is put for-ward by the manufacturer to show that conducting such studies isunnecessary. For a preventive vaccine, reproductive toxicity assess-ments are generally restricted to prenatal and postnatal developmen-tal studies, because the primary concern is any potential untowardeffect on the developing embryo, fetus or newborn. The need toconduct fertility and post-weaning assessments should be consideredon a case-by-case basis. The animal model chosen should develop

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an immune response to the vaccine, which is usually determined byserum antibody measurements. In addition, it is important to evaluatematernal antibody transfer by measuring vaccine-induced antibodyin cord or fetal blood to verify exposure of the embryo or fetus tomaternal antibody. The route of administration should mimic theclinical route of administration. Ideally, the maximal human doseshould be administered to the test animal. If it is not possible toadminister the full human dose, e.g. limitations on the total volumethat can be administered, or if local toxicity is observed that mayresult in maternal stress, a dose that exceeds the human dose on amg/kg basis and is able to induce an immune response in the animalshould be used.

To assess any potential adverse effects of the vaccine during theperiod of organogenesis, the gestating animal is usually exposed tothe vaccine during the period from implantation until closure of thehard palate and end of gestation defined as stages C, D and E in theICH S5a document (27). Because of the relatively short gestationperiod of most animal models used, pre-mating treatment is fre-quently required to ensure maximal exposure of the embryo or fetusto the vaccine-induced immune response. For a preventive vaccine,the number of doses administered depends on the time of onset andduration of the response. Booster immunizations may be necessary atcertain times during the period of gestation to maintain a high level ofantibody throughout the gestation period and to expose the develop-ing embryo to the components of the vaccine formulation. End-pointsinclude, but are not limited to, viability, resorptions, abortions, fetalbody weight and morphology. The reader is referred to other publica-tions for guidance on end-points used to evaluate potential toxiceffects of the product on development of the embryo or fetus (27). Itis also recommended that a period of postnatal follow-up of pupsfrom birth to weaning be incorporated in the study design to assessnormality of growth, body weight gain, suckling activity and viability.Studies should therefore be designed so that test groups are dividedinto subgroups. Half of the animals should be delivered by Caesareansection and the other half allowed to deliver their pups without surgi-cal intervention.

4.2.3 Genotoxicity and carcinogenicity studiesGenotoxicity studies are normally not needed for the final vaccineformulation. However, they may be required for particular vaccinecomponents such as novel adjuvants and additives. If needed, the invitro tests for mutations and chromosomal damage should be doneprior to first human exposure. The full battery of tests for genotoxicitymay be performed in parallel with clinical trials (28).

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Carcinogenicity studies are not required for vaccine antigens. How-ever, they may be required for particular vaccine components such asnovel adjuvants and additives.

4.2.4 Safety pharmacologyThe purpose of safety pharmacology is to investigate the effects of thecandidate vaccine on vital functions. If data from nonclinical and/orhuman clinical studies suggest that the vaccine (e.g. one based onspecific toxoids) may affect physiological functions (e.g. centralnervous system, respiratory, cardiovascular and renal functions)other than those of the immune system, safety pharmacology studiesshould be incorporated into the toxicity assessment. Useful informa-tion on this topic can be found in the Note for Guidance on safetypharmacology studies for human pharmaceuticals (29).

4.2.6 Pharmacokinetic studiesPharmacokinetic studies (e.g. for determining serum or tissue concen-trations of vaccine components) are normally not needed. The needfor specific studies should be considered on a case-by-case basis (e.g.when using novel adjuvants or alternative routes of administration)and may include local deposition studies that would assess the reten-tion of the vaccine component at the site of injection and its furtherdistribution (e.g. to the draining lymph nodes). Distribution studiesshould be considered in the case of new formulations, novel adjuvantsor when alternative routes of administration are intended to be used(e.g. oral or intranasal).

5 Special considerations5.1 Adjuvants

Adjuvants may be included in vaccine formulations or co-administered with vaccines to enhance the immune responses to par-ticular antigen(s), or to target a particular immune response. It isimportant that the adjuvants used comply with pharmacopoeial re-quirements where they exist, and that they do not cause unacceptabletoxicity.

Adjuvant activity is a result of many factors and the immune responseobtained with one particular antigen/adjuvant formulation cannot, asa rule, be extrapolated to another antigen. Individual antigens vary intheir physical and biological properties and antigens may interactdifferently with an adjuvant. Adjuvants must be chosen according tothe type of immune response desired and they must be formulatedwith the antigen in such a way that distribution of both is optimized toensure availability to the relevant lymphatic tissues. The route of

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administration of the vaccine is also an important factor influencingthe efficacy and safety of an adjuvant.

The effect of the adjuvant should be demonstrated in preclinicalimmunogenicity studies. If no toxicological data exist for a new adju-vant, toxicity studies of the adjuvant alone should first be performed.In general, assessment of new or novel adjuvants should be under-taken as required for new chemical entity (30–32). These data may beobtained by the vaccine manufacturer or by the producer of theadjuvant. In addition to assessing the safety of the adjuvant by itself itis also important to assess whether the combination of antigen andadjuvant exerts a synergistic adverse effect in the animal model(33, 34). When species-specific proteins (e.g. cytokines) are used asnovel adjuvants, the issue of species-specific response should beconsidered.

When evaluating the safety profile of the combination of adjuvantand vaccine, the formulation proposed for clinical use should beused.

Compatibility of the adjuvant(s) (e.g. lack of immune interference)with all antigenic components present in the vaccine should beevaluated.

If applicable, adsorption of all antigenic components present in thevaccine should be shown to be consistent on a lot-to-lot basis. Poten-tial desorption of antigen during the shelf-life of the product shouldbe performed as a part of stability studies, the results reported andspecifications set, as this may affect not only immunogenicity, but alsothe toxicity profile of the product.

It should be noted that no adjuvant is licensed in its own right, butonly as a component of a particular vaccine.

5.2 Additives (excipients and preservatives)

Where a new additive is to be used, for which no toxicological dataexist, toxicity studies of the additive alone should first be performedand the results documented according to the guidelines for newchemical entities (31). The compatibility of a new additive with allvaccine antigens should be documented together with the toxicologi-cal profile of the final vaccine formulation under consideration inanimal models as outlined in section 4.

5.3 Vaccine formulation and delivery device

The vaccine formulation (i.e. liquid form, capsules or powder), aswell as the delivery device, may have an impact on the uptake of

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the vaccine, its effectiveness and safety. Ideally, the delivery deviceand vaccine formulation tested in an animal safety study should beidentical to those intended to be used clinically. However, animalmodels in which delivery devices intended for clinical use can betested may not be available. In these instances, in order to develop anappropriate animal model, it may be necessary to conduct pilot stud-ies to define and optimize the conditions for drug delivery in theanimal model before it can be used to assess the preclinical safety ofthe product.

5.4 Alternative routes of administration

When using a vaccine formulation administered by alternative routes(e.g. intranasal, oral, intradermal, rectal and intravaginal routes), itcan be assumed that their potency, relevant immunogenicity, toler-ability, toxicity, and long-term safety may differ from that of productsdelivered by the parenteral route. Thus, when different routes ofadministration are proposed, nonclinical safety studies may have tobe conducted using vaccine formulation and/or adjuvant alone in asuitable animal model to address the specific safety concerns associ-ated with vaccine administration by these routes. Particular issuesrelevant to vaccines administered using alternative routes that mayneed to be considered are discussed below.

5.4.1 Animal modelsA special consideration for vaccines administered by alternativeroutes should be the anatomy and physiology of the site of vaccineadministration of the particular animal model chosen and its accessi-bility for the administration of the vaccine. For example, for intra-nasally administered products, the species chosen should ideally bereceptive to spray administration of the product. In general, rabbitsand dogs are useful test models for use of spray devices; however,their olfactory bulbs are highly protected and special techniqueswould be required to ensure that the test product reached this organ.Although mice and rats are useful models, intranasal administrationto these species presents technical difficulties. Intranasal administra-tion to non-human primates may be preferable, if they are susceptibleto the infectious agent in question.

Depending on the level of concern regarding a particular route ofadministration or when there are species-specific differences betweenthe animal models in their sensitivity to the candidate vaccine, it maybe necessary to address the preclinical safety of the product in morethan one safety study and in more than one animal model.

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5.4.2 DoseAs the optimal dose derived from studies using the parenteralroute of administration may differ from the dose used for alternativeroute(s) of administration, dose-finding studies may need to be con-ducted for a particular route of administration. Also, considerationshould be given to the total volume of the vaccine administered as itmay affect the outcome of the safety study. For example, intranasaladministration of more than 5ml of test preparation per nostril to amouse would result in the test preparation being swallowed, ratherthan being adsorbed by the nasal mucosa.

5.4.3 End-pointsThe toxicity end-points would include those described in section 4 andmay include additional outcome measures that would depend on theroute of administration and specific concerns associated with the par-ticular route and target organ. For example, if there is concern aboutthe potential passage of vaccine components to the brain followingintranasal administration, immunohistology and “in situ” methodsand/or neurological assays and examinations may be necessary. Forvaccines administered by inhalation, outcome measures may includepulmonary function tests and data on histopathology of the lungs.Considerable efforts may be required to develop appropriate meth-ods to address potential safety concerns associated with the use ofnew routes of administration.

5.4.4 Immunogenicity assessmentThe development of appropriate assays for measuring mucosal im-mune responses is critical for vaccines that are expected to function asmucosal immunogens because serological assays alone may not reflectthe relevant immune response for a mucosal vaccine. Thus, in addi-tion to measuring serological responses, it may be necessary to evalu-ate T cell responses, antibody-secreting cells and cytokine production.In addition, assays may need to be developed to assess the inductionof local and systemic responses at sites distant from administration ofthe vaccine antigen.

6 Specific considerations for particular types of vaccines

In addition to the testing strategies outlined in sections 3, 4 and 5,studies may be necessary to address specific safety concerns associ-ated with particular product types using suitable in vitro and in vivotest methods. The specific testing requirements for live attenuatedand combination vaccines are discussed below. Detailed informationregarding the production and control of other types of vaccine isavailable in the WHO guidance documents for production and con-

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trol (13), and should be consulted. For example, in the recently devel-oped guidelines for DNA (16) and synthetic peptide vaccines (18, 35),as well as for particular vaccines such as Hib conjugated vaccine (26),the issues relevant for nonclinical testing are discussed and should beconsidered in the development of an appropriate design for thenonclinical study of the vaccine in question.

6.1 Live attenuated vaccines

An assessment of the degree of attenuation, and the stability ofthe attenuated phenotype, are important considerations for thenonclinical testing programme of a live attenuated vaccine. Labora-tory markers of attenuation are invaluable for this purpose. Thesemarkers should be capable of distinguishing the attenuated vaccinefrom fully virulent wild-type strains and, ideally, of detecting partialreversion to full virulence. To assess the stability of the attenuationphenotype, the vaccine may be passaged under production conditionsbeyond the maximum passage number to be used for production.Stability of attenuation may also be assessed by passage under condi-tions that are outside the conditions to be used for vaccine produc-tion. For example, higher or lower temperatures may exert selectionpressure for reversion to virulence. The marker(s) of attenuation maysubsequently be used to qualify new vaccine seed preparations and tomonitor the effect of any significant changes in production conditionsof the attenuated phenotype.

If the wild-type organism is neurotropic, or if passages through neuraltissue have been used in the attenuation of a virus vaccine, then a testfor neurovirulence should be performed at least at the level of thevaccine seed. A neurovirulence test is not necessarily required forall live attenuated vaccines. The specifications for an appropriateneurovirulence test depend on the organism under test and shouldbe capable of distinguishing the attenuated vaccine from fully viru-lent wild-type strains and, ideally, of detecting partial reversion tofull virulence. Specific reference preparations may be needed forthis purpose. Neurovirulence tests in small animal models may beacceptable.

If the live attenuated vaccine is based on a genetically modified organ-ism, then an environmental risk assessment may be required as part ofthe preclinical evaluation. An investigation into the possible sheddingof vaccine organisms following administration contributes to the envi-ronmental risk assessment. For all live attenuated vaccines, infor-mation on the likelihood of exchange of genetic information withnon-vaccine strains may be required and suitable nonclinical testsmay be designed to provide data for this purpose.

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6.2 Combined vaccines

New combinations produced either by formulation or at the time ofreconstitution of antigens or serotypes should be studied for ap-propriate immunogenicity in an animal model, if available, beforeinitiation of human clinical trials (36, 37). Combined antigens shouldbe examined by appropriate physicochemical means to evaluatepossible changes to antigen properties on combination, such as degreeof adsorption to aluminium adjuvants, as well as stability of thecombination.

The immune response to each of the antigens in the vaccine should beassessed, including the quality of response and any potential interfer-ence and incompatibilities between combined antigens. It is prefer-able to study a new combination in comparison with the individualantigens in animals to determine whether augmentation or diminu-tion of response occurs.

The need to evaluate the safety of the new combination in an animalmodel should be considered on a case-by-case basis. Such evaluationis likely to be necessary if there is concern that combining antigensand/or adjuvants may lead to problems of toxicity (e.g. noveladjuvant).

Similar consideration for nonclinical testing will also apply to caseswhere a new candidate single-component vaccine is developed froman already licensed combined vaccine (e.g. monovalent oral poliovaccine versus trivalent oral polio vaccine).

AuthorsThe first draft of these Guidelines on preclinical evaluation of vaccines was pre-pared by the members of the WHO drafting group following the meeting held at theNational Institute of Public Health and the Environment (RIVM), the Netherlands14–15 March 2002 attended by:

Dr M. Gruber, Scientific Reviewer, Division of Vaccines and Related ProductsApplication, Center for Biologics Evaluation and Research, Food and Drug Admin-istration, Rockville, MD, USA; Dr A. Homma, Bio-Manguinhos Oswaldo Cruz Foun-dation, Rio de Janeiro, Brazil; Dr J.G. Kreeftenberg, Bureau for InternationalCooperation, National Institute of Public Health and the Environment, Bilthoven, theNetherlands; Dr J.W. van der Laan, National Institute of Public Health and theEnvironment, Bilthoven, the Netherlands; Dr E. Griffiths, Coordinator, QualityAssurance and Safety of Biologicals, World Health Organization, Geneva, Switzer-land; Dr I. Knezevic, Quality Assurance and Safety of Biologicals, World HealthOrganization, Geneva, Switzerland.

A second draft was prepared following a discussion on special immunologicalconsiderations held at Agence Française de Sécurité Sanitaire des Produits de

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Santé, Lyon, France, 17 June 2002 attended by the following: Dr F. Fuchs, AgenceFrançaise de Sécurité Sanitaire des Produits de Santé, Lyon, France; Dr D. Masset,Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS), StDenis, France; Dr C. Ratignier Agence Française de Sécurité Sanitaire desProduits de Santé, St Denis, France; Dr Marc Pallardy, Agence Française deSécurité Sanitaire des Produits de Santé (AFSSAPS), St Denis, France, and afurther meeting of drafting group held in Geneva from 1–2 July 2002.

A third draft was prepared by the drafting group after an informal WHO Consulta-tion on preclinical evaluation of vaccines: regulatory expectations, held in Genevafrom 12–13 December 2002, attended by the following participants:

Dr T. Bektimirov, Deputy Director, Tarasevic State Research Institute for Standard-ization and Control of Medical Biological Preparations, Moscow, Russian Federa-tion; Dr E. Chaves Leal, Vice Director, Instituto Nacional de Control de QualidadSaude, Rio de Janeiro, Brazil; Dr W. Egan, Acting Director, Office of VaccinesResearch and Review, Center for Biologics Evaluation and Research, Food andDrug Administration, Rockville, MD, USA; Dr E. Griffiths, Associate Director General,Biologics and Genetic Therapies, Health Canada, Ottawa, Canada; Dr M. Gruber,Scientific Reviewer, Division of Vaccines and Related Products Application, Centerfor Biologics Evaluation and Research, Food and Drug Administration, Rockville,MD, USA; Dr M. Haase, Paul Ehrlich Institute, Langen, Germany; Dr A. Homma,Developing Country Vaccine Manufacturer’s Network, c/o Bio-ManguinhosOswaldo Cruz Foundation, Rio de Janeiro, Brazil; Dr J.G. Kreeftenberg, Bureau forInternational Cooperation, National Institute of Public Health and the Environment,Bilthoven, the Netherlands; Dr J.W. van der Laan, National Institute of Public Healthand the Environment, Bilthoven, the Netherlands; Dr R. Leke, Department ofImmunology and Microbiology, Faculty of Medicine, University of Yaounde,Yaounde, Cameroon; Dr Lei Dianliang, Deputy Director, National Institute for theControl of Pharmaceutical and Biological Products, Beijing, People’s Republic ofChina; Dr Lazara Martínez Muñoz, Centro para el Control Estatal de la Calidad de losMedicamentos, Havana, Cuba; Dr D. Masset, Agence française de SécuritéSanitaire des Produits de Santé, Saint-Denis, France; Dr R. Mignolet, RLM Consult-ing, Wavre, Belgium; Dr P. Minor, Head, Division of Virology, National Institute forBiological Standards and Control, Potters Bar, Herts., England; Dr G. Orefici, IstitutoSuperiore di Sanità, Rome, Italy; Dr M. Pallardy, Agence Française de SécuritéSanitaire des Produits de Santé, Veille Toxicologique/Vigilance, St Denis, France; DrJ. Petricciani, Carlsbad, USA; Dr P. Pitisuttithum, Vaccine Trial Centre, Faculty ofTropical Medicine, Mahidol University, Bangkok, Thailand; Dr F. Reigel, Vice Direc-tor, Swissmedic, Biological Medicines and Laboratories, Berne, Switzerland; Dr J.Robertson, National Institute for Biological Standards and Control, Potters Bar,Herts., England; Dr L. Slamet, Deputy for Therapeutic Products, Narcotic, Psycho-tropic and Addictive Substance Control, Directorate General of Food and DrugControl, Ministry of Health, Jakarta, Indonesia; Dr A.K. Tahlan, Central DrugsLaboratory, Central Research Institute, Kasauli, India; Ms C. Chamberlin, ScientificSecretary of European Pharmacopoeia Group of Experts on Vaccines, Strasbourg,France; Dr B. Meignier, IABS c/o Director, External R & D, Aventis Pasteur SA, Marcyl’Etoile, France; Dr B.J. Ledwith, Director, Biologics Safety Assessment, MerckResearch Laboratories, West Point, PA, USA; Dr F. Verdier, Head, Product SafetyAssessment, Aventis Pasteur, Marcy l’Etoile, France; Dr G. del Giudice, Head,Animal Models and Serology, Research Center, Chiron SpA, Siena, Italy.

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WHO secretariat: Dr M.P. Kieny, Director, Initiative to Vaccine Research; Dr L.Rago, Coordinator, Quality Assurance and Safety of Medicines; Dr D. Wood,Acting Coordinator, Quality Assurance and Safety of Biologicals; Mr L. Belgharbi,Access to Technologies; Dr N. Dellepiane, Access to Technologies; Dr P. Duclos,Vaccine Assessment and Monitoring; Dr D. Kioy, Tropical Disease Research; DrI. Knezevic, Quality Assurance and Safety of Biologicals; Dr E. Uramis Diaz,Access to Technologies; Dr S. Osmanov, Initiative to Vaccine Research/HVI.

The final draft (WHO/BS/03.1969) was prepared by Dr E. Griffiths, Dr M. Gruber,Dr D. Masset, Dr F. Verdier, Dr D. Wood and Dr I. Knezevic, following a meetingheld in Geneva, 9–10 June 2003, and taking into account comments made by theExpert Committee on Biological Standardization at its meeting in February 2003 aswell as comments made by the reviewers of the document.

References1. WHO guidelines for clinical evaluation of vaccines: regulatory expectations.

In: WHO Expert Committee on Biological Standardization. Fifty-secondReport. Geneva, World Health Organization, 2004 (WHO Technical ReportSeries, No. 924 Annex 1).

2. Biological standardization and control. A scientific review commissioned bythe UK National Biological Standards Board. Geneva, World HealthOrganization, 1997 (WHO/BLG/97.1).

3. Biotechnology and world health. Risks and benefits of vaccines andother medical products produced by genetic engineering. Proceedings ofa WHO meeting. Geneva, World Health Organization, 1997 (WHO/VRD/BLG/97.01).

4. WHO Manual of laboratory methods for testing vaccines used in the WHOExpanded Programme on Immunization. Geneva, World HealthOrganization, 1997, Annex 1 (WHO/VSQ/97.04).

5. World Health Organization. Laboratory biosafety manual, 2nd ed. (revised)Geneva, World Health Organization, 2003.

6. OECD principles on Good Laboratory Practice (revised 1997). Paris,Organisation for Economic Co-operation and Development, 1997 (ENV/MC/CHEM (98) 17).

7. Good manufacturing practices for biological products. In: WHO ExpertCommittee on Biological Standardization. Forty-second Report. Geneva,World Health Organization, 1992 (WHO Technical Report Series, No.822):20–30.

8. Good manufacturing practice: supplementary guidelines for the manufactureof the investigational pharmaceutical products for clinical trials in humans.In: WHO Expert Committee on Specifications for PharmaceuticalPreparations: Thirty-Fourth Report. Geneva, World Health Organization,1996, Annex 7 (WHO Technical Report. Series, No. 863).

9. Good manufacturing practices for pharmaceutical products. In: WHO ExpertCommittee on Specifications for Pharmaceutical Preparations. Thirty-second

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Report. Geneva, World Health Organization, 1992 Annex 1 (WHO TechnicalReport Series, No. 823).

10. Note for guidance on comparability of medicinal products containingbiotechnology-derived proteins as drug substance. London, Committee forProprietary Medicinal Products, 2000 (CPMP/BWP/3207/00).

11. European Commission Regulations No. 541/95, 542/95, 1146/98 and 1069/98.

12. Griffiths E. Efficacy of whole-cell pertussis vaccine. In: Wardlaw AC, PartonR, eds. Pathogenesis and immunity in pertussis. Chichester, Wiley, 1988:pp. 353–374.

13. Recommendations and guidelines for biological substances used inmedicine and other documents. Geneva, World Health Organization, 2002(WHO Technical Report Series, No. 910):99–102.

14. Requirements for the use of animal cells as in vitro substrates for theproduction of biologicals. In: WHO Expert Committee on BiologicalStandardization. Forty-seventh Report. Geneva, World Health Organization,1998, Annex 1 (WHO Technical Report Series, No. 878).

15. Guidelines on transmissible spongiform encephalopathies in relation tobiological and pharmaceutical products. Geneva, World HealthOrganization, 2003 (WHO/BCT/QSD/03.01).

16. Guidelines for assuring quality of DNA vaccines. In: WHO ExpertCommittee on Biological Standardization. Forty-seventh Report. Geneva,World Health Organization, 1998, Annex 3 (WHO Technical Report Series,No. 878).

17. Guidelines for assuring the quality of pharmaceutical and biologicalproducts prepared by recombinant DNA technology. In: WHO ExpertCommittee on Biological Standardization. Forty-first Report. Geneva,World Health Organization, 1991, Annex 3 (WHO Technical Report Series,No. 814).

18. Guidelines for the production and quality control of synthetic peptidevaccines. In: WHO Expert Committee on Biological Standardization.Forty-eighth Report. Geneva, World Health Organization, 1999, Annex 1(WHO Technical Report Series, No. 889).

19. Guidance for industry: content and format of chemistry, manufacturing andcontrols information and establishment description information for a vaccineor related product. Federal Register, 1999, 2:518–519 (Center for BiologicsEvaluation and Research, US Food and Drug Administration).

20. Guidelines for national authorities on quality assurance for biologicalproducts. In: WHO Expert Committee on Biological Standardization.Forty-second Report. Geneva, World Health Organization, 1992 (WHOTechnical Report Series, No. 822):31–46.

21. Regulation and licensing of biological products in countries with newlydeveloping regulatory authorities. In: WHO Expert Committee on BiologicalStandardization. Forty-fifth Report. Geneva, World Health Organization, 1995(WHO Technical Report Series No. 858):21–35.

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22. Scientific considerations for the regulation and clinical evaluation of HIV/AIDS preventive vaccines: report from a WHO–UNAIDS Consultation 13–15March 2001, Geneva Switzerland. AIDS, 2002, 16:W15–W25.

23. Note for guidance on repeated dose toxicity. London, Committee forProprietary Medicinal Products, 1999 (CPMP/SWP/1042/99).

24. Note for guidance on non-clinical local tolerance testing of medicinalproducts. London, Committee for Proprietary Medicinal Products, 2000(CPMP/SWP/2145/00).

25. Wraith DC, Goldman M, Lambert PH. Vaccination and autoimmune disease:what is the evidence? Lancet, 2003, 362:1659–1666.

26. Recommendations for Haemophilus influenzae type b conjugate vaccines.In: WHO Expert Committee on Biological Standardization. Forty-ninth Report.Geneva, World Health Organization, 2000 (WHO Technical Report Series,No. 897).

27. Note for guidance for reproductive toxicology: detection of toxicity toreproduction for medicinal products. London, Committee for ProprietaryMedicinal Products (CPMP/ICH/386/95).

28. Note for guidance on genotoxicity: a standard battery for genotoxicitytesting of pharmaceuticals. London, Committee for Proprietary MedicinalProducts, 1995 (CPMP/ICH/174/95).

29. Note for guidance on safety pharmacology studies for humanpharmaceuticals. London, Committee for Proprietary Medicinal Products(CPMP/ICH/539/00).

30. ICH M3(M) Nonclinical safety studies for the conduct of human clinical trialsfor pharmaceuticals. London, Committee for Proprietary Medicinal Products,2000 (CPMP/ICH/286/95 modification).

31. Guidance for industry: nonclinical studies for development ofpharmaceutical excipients. Draft. September 2002.

32. Note for guidance on excipients, antioxidants and antimicrobialpreservatives in the dossier for application for marketing authorisation of amedicinal product. London, Committee for Proprietary Medicinal Products,2003 (CPMP/QWP/419/03).

33. Note for guidance on preclinical pharmacological and toxicological testingof vaccines. London, Committee for Proprietary Medicinal Products, 1998(CPMP/SWP/465/95).

34. Goldenthal KL, Cavagnaro JA, Alving CR, Vogel FR. Safety evaluation ofvaccine adjuvants: National Cooperative Vaccine Development MeetingWorking Group. AIDS Research and Human Retroviruses, 1993, 9(suppl1):S47–S51.

35. Guidance for industry for the submission of chemistry, manufacturing, andcontrols information for synthetic peptide substances. Center for DrugEvaluation and Research and Center for Biologics Evaluation and Research,1994.

36. Verdier F, Patriarca C, Descotes J. Autoantibodies in conventional toxicitytesting. Toxicology, 1997, 119:51–58.

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37. Guidance for industry for the evaluation of combination vaccines forpreventable diseases: production, testing and clinical studies. US Food andDrug Administration, 1997.

38. Note for guidance on pharmaceutical and biological aspects of combinedvaccines. London, Committee for Proprietary Medicinal Products, 1999(CPMP/BWP/477/97). Geneva, World Health Organization, 2003.

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AppendixList of tissues to be collected in a repeated dosetoxicity study

adrenal glands

aorta

bone (femur) and articulation

bone (sternum) with bone marrow

bone marrow smears1

brain

bronchi (main-stem)

caecum

colon

duodenum

epididymides

eyes

heart

ileum

injection site(s) (a sample should be taken from the area of injection)

jejunum

kidneys and ureters

larynx

liver

lungs

lymph node (mandibular)

lymph node (mesenteric)

mammary gland

oesophagus

optic nerves

1 Bone marrow smears should be prepared at the scheduled necropsy for all animalsincluding any moribund animals killed during the study. The smears should be fixed inmethanol and then stained by the May-Grunwald-Giemsa method.

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ovaries and oviducts

pancreas

parathyroid glands

Peyer’s patches

pituitary gland

prostate

rectum

salivary glands (mandibular, parotid, sublingual)

sciatic nerves

seminal vesicles

skeletal muscle

skin

spinal cord (cervical, thoracic, lumbar)

spleen

stomach

testes

thymus

thyroid glands

tongue

trachea

ureters

urinary bladder

uterus (horns + cervix)

vagina

all gross lesions

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 2Recommendations for the production and control ofpneumococcal conjugate vaccines

Recommendations published by WHO are intended to be scientific andadvisory in nature. The parts of each section printed in type of normalsize have been written in a form, such that, should a national regulatoryauthority so desire, they may be adopted as they stand as definitivenational requirements or used as the basis of such requirements. Thoseparts of each section printed in small type are comments and recommen-dations for guidance for those manufacturers and national regulatoryauthorities which may benefit from additional information.

It is recommended that modifications be made only on condition that themodifications ensure that the vaccine is at least as safe and efficaciousas that prepared in accordance with the recommendations set out below.

The terms “national regulatory authority” and “national control laboratory”as used in these recommendations, always refer to the country in whichthe vaccine is manufactured.

Introduction 66

General considerations 66

Special considerations 68

Combined vaccines containing pneumococcal polysaccharide conjugatecomponents 69

Part A. Manufacturing recommendations 70A.1 Definitions 70A.2 General manufacturing recommendations 71A.3 Production control 72A.4 Records 84A.5 Retained samples 84A.6 Labelling 84A.7 Distribution and transport 84A.8 Stability, storage and expiry date 84

Part B. Requirements for national regulatory authorities 86B.1 General 86B.2 Official release and certification 86B.3 Reactogenicity and immunogenicity of vaccine in humans 86

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Authors 87

References 88

AppendixSerological criteria for evaluation and licensure of newpneumococcal conjugate vaccine formulations for use in infants 92

Appendix

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Introduction

Recommendations (formerly known as Requirements) for pneumo-coccal polysaccharide vaccines were drafted in 1980 but were neveradopted by the WHO Expert Committee on Biological Standardiza-tion (1). Vaccines based on the capsular polysaccharides of the 23serotypes of Streptococcus pneumoniae most commonly associatedwith human disease have been licensed in many countries (2). Thesevaccines have been shown to be efficacious against invasive pneumo-coccal disease and have proved to be effective for the protection ofindividuals who are at particular risk of infection. Nevertheless, theirinability to elicit protective responses in young infants or to inducegood immunological memory has prevented their inclusion in na-tional infant immunization schedules.

The development of bacterial capsular polysaccharide–proteinconjugates represents a major advance in prophylaxis against bac-terial infections (3). Following the successful introduction of theHaemophilus influenzae type b conjugate (Hib) and meningococcal Cconjugate (MenC) vaccines into paediatric vaccination schedules,considerable progress has been made in the development of similarconjugate vaccines based on pneumococcal capsular polysaccharides.Glycoconjugate vaccines are both physically and immunobiologicallydistinct from their unconjugated counterparts, emphasizing the needfor recommendations specifically for these products.

General considerations

Infections caused by S. pneumoniae, the pneumococcus, are respon-sible for substantial morbidity and mortality, particularly in the veryyoung and in the elderly (2, 4, 5). Pneumococci are grouped intoserotypes on the basis of their chemically and serologically distinctcapsular polysaccharides. Of the 90 pneumococcal serotypes (6), thecapsular polysaccharides of the 23 most commonly associated withdisease are included in the polysaccharide vaccines produced by vari-ous manufacturers. These vaccines are effective in individuals fromabout 2 years of age but, as they elicit T-cell independent immunity,they are not effective in younger children. In addition, they fail toinduce boostable immunity and have little or no impact on nasopha-ryngeal carriage (7). In contrast, polysaccharide–protein conjugateshave been shown to be highly immunogenic in infants and to induceT-cell dependent immunity. Several pneumococcal conjugate vac-cines are now available or are at an advanced stage of development(8–10). The results of controlled clinical trials of these vaccines have

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demonstrated that such conjugates are both safe and highly immuno-genic, T-cell dependent antigens (11, 12). They have been shown toinduce high levels of serum antibody and to offer protective immunityagainst invasive pneumococcal disease (13). They are effective inyoung children, induce immunological memory and reduce nasopha-ryngeal carriage of the pneumococcal serotypes included in the for-mulation (14). A 7-valent conjugate, manufactured using diphtheriaprotein CRM197 as the carrier protein for all seven serotypes, wasfirst licensed in the USA in 2000 and has become increasingly avail-able worldwide.

Protective levels of antibody elicited by the CRM197 conjugatedvaccines against invasive pneumococcal disease have been estimatedusing the data from three clinical efficacy trials: one in NorthernCalifornia; one among Navajo Indians and one in Soweto, SouthAfrica. The aggregate efficacy for the seven serotypes these vaccineshad in common was 93.0% (95% confidence interval, 81.0–98.2%).Using the data from enzyme-linked immunosorbent assays (ELISA)for anti-capsular polysaccharide antibody, an estimate of 0.35mg/mlaggregated across the serotypes was associated with the point-estimate of clinical efficacy against invasive disease (see Appendixfor additional details). However, this reference value is neitherapplicable to the determination of the protective status of the indi-vidual nor to protection against other disease end-points, e.g. pneu-monia or otitis media. Practical or ethical considerations may makeit impossible to perform protective efficacy trials of most new vaccineformulations. Therefore, this reference value will be important forthe licensure of future products using data from immunogenicitytrials.

Differences in the incidence of serotypes causing disease from onecontinent to another have led to the development of pneumococcalvaccine formulations consisting of increasing numbers of conjugatedcomponents (15). Recently clinical trials of 7-valent and 9-valentformulations have been completed in Finland and South Africa re-spectively (16–18), and further formulations with potentially greatercoverage are under development (9). From a practical perspective,however, it is evident that there is a limit to the number of serotypesthat can be included in such conjugate vaccine formulations and theincidence of disease-causing serotypes in the target population shouldbe taken into consideration before vaccine development. Althoughgeographical and temporal factors undoubtedly contribute to differ-ences in the incidence rates between regions, the impact of differ-ences between national epidemiological surveillance systems on caseascertainment may also prove to be a critical factor in the assessment

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of pneumococcal vaccine coverage (19). The serotype composition ofpneumococcal vaccines should be agreed with the national regulatoryauthority based on appropriate epidemiological data on the targetpopulation. The superiority of a vaccine should not be assumed on thebasis of the number of serotypes included unless there is evidence thatthe inclusion of additional serotypes is likely to enhance its effective-ness in a particular epidemiological setting.

Special considerations

The production and control of conjugate vaccines is more complexthan that of their unconjugated capsular polysaccharide counterparts.Polysaccharide vaccines consist of defined chemical substances that,if prepared to the same specifications, can reasonably be expectedto have comparable potencies. Although only the 7-valent conjugateformulation has been licensed to date, experience with H. influenzaetype b and meningococcal conjugate vaccines suggests that effectivepneumococcal vaccines may be developed that differ both in thenature of the saccharide and the carrier protein employed. Vaccinesare under development that utilize carrier proteins other thanCRM197 and vaccine formulations could be developed in which morethan one carrier is employed. The manufacturer has a choice of pos-sible carrier proteins providing that the resulting vaccine is safe andelicits a T-cell dependent, protective immune response.

Unfortunately, the lack of a suitable animal model for all pneumococ-cal serotypes makes it impossible to assess the potency of these vac-cines for humans on the basis of studies in animals. Consequently, itis important that new pneumococcal vaccine formulations are evalu-ated in humans for immunogenicity by monitoring the productionof serotype-specific immunoglobulin G (IgG). Immune responses topneumococcal vaccines have been measured using methods thatdetermine either the total amount of antibody binding to capsularpolysaccharide or the amount of functional antibody present in se-rum. Antibody binding is typically evaluated by the use of ELISAs orradioimmunoassays (20–22a), whereas the opsonophagocytic assay isused to measure functional antibodies (23–25). Clinical studies ofconjugate vaccines have shown a good association between antibodylevels measured by ELISA and protection (see Appendix). However,such studies also usually include an analysis of a subset of sera toconfirm their functional (e.g. opsonophagocytic) activity. Whicheverassay is used, it should be standardized so as to ensure comparabilityof data both between laboratories and between different clinical

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studies. A set of calibration sera is available to help establish compa-rability between laboratories (26). As conjugate vaccines should in-duce a T-cell dependent immune response, this should also beevaluated during clinical trials. Indicators of T-cell dependent immu-nity include the production of predominantly high-avidity IgG anti-body and the demonstration of a good booster response in childrenwho have already had the primary vaccination (14).

Given the lack of a suitable animal model that will predict the potencyof all pneumococcal serotypes, the strategy for the control of thevaccine is dominated by the use of tests for molecular characterizationand purity. These tests focus on physicochemical criteria to ensureeach vaccine lot is consistent with the specification of the vaccine lotsused in the definitive clinical trials that confirmed their safety andefficacy. Animal studies form an essential part of the development ofthese vaccines to provide evidence that they induce T-cell dependentimmunity and to characterize the immunogenicity of the vaccine dur-ing stability studies. However, an immunogenicity test in animals isnot necessary for routine lot release when vaccine consistency hasbeen assured by alternative means.

Combined vaccines containing pneumococcalpolysaccharide conjugate components

The introduction of Hib and MenC conjugates as additional elementsof infant immunization programmes has served to highlight the needto combine paediatric vaccines for effective vaccine delivery (27).Vaccine formulations with multiple components that include pneu-mococcal conjugates are likely to be developed within the next de-cade. If one or more conjugated pneumococcal components areindicated for co-administration with other vaccines, the possible ef-fects on the clinical performance of each component in the vaccine,including the pneumococcal conjugate components, should be evalu-ated in terms of their safety and immunogenicity. Similarly, the clini-cal effect of concomitant administration of a pneumococcal conjugatevaccine with other vaccines at different sites should be evaluated.Because of the problems associated with performing physicochemicalanalyses on complex vaccine formulations, the manufacturer shouldconsider which batch release tests are appropriate to perform on finalbulks of a particular product and which tests should be performed onfinal lots of such vaccines. The tests should be agreed with thenational regulatory authority.

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Part A. Manufacturing recommendations

A.1 DefinitionsA.1.1 Proper name

The proper name of the vaccine should be “Pneumococcal conjugatevaccine” translated into the language of the country of use. Theserotypes included in the vaccine should be associated with the nameof the vaccine and listed in the packaging material. The use of thisproper name should be limited to vaccines that satisfy the recommen-dations formulated below.

A.1.2 Descriptive definition

Multivalent pneumococcal conjugate vaccine is a preparation ofcapsular polysaccharide from specific serotypes of Streptococcuspneumoniae that are covalently linked to carrier protein.

A.1.3 International reference materials

No formally established international reference materials that wouldallow the standardization of immune responses to pneumococcal con-jugate vaccines are currently available.

The following reagents are available through the courtesy ofindividuals, manufacturers and national regulatory or referencelaboratories:

C-polysaccharide (Statens Serum Institute, Copenhagen, Denmark)Capsular polysaccharides (American Type Culture Collection(ATCC), Manassas, Virginia, USA)89-SF reference serum (Dr Carl Frasch, Center for Biologics Evalua-tion and Research, US Food and Drug Administration (CBER/FDA), Rockville, MD, USA) (22)96DG secondary reference serum (provided by Dr David Goldblattand distributed by National Institute for Biological Standards andControl (NIBSC), Potters Bar, Herts., England)ELISA calibration sera (provided by Dr David Goldblatt and distrib-uted by NIBSC, Potters Bar, Herts., England) (26)Pneumococcal serotyping reagents (Statens Serum Institute,Copenhagen, Denmark)HL-60 cells (ATCC, Manassas, Virginia, USA)

A.1.4 Terminology

Master seed lot. A bacterial suspension of S. pneumoniae derived froma strain that has been processed as a single lot and is of uniformcomposition. It is used for the preparation of the working seed lots.

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Master seed lots shall be maintained in the freeze-dried form or befrozen below -45°C.

Working seed lot. A quantity of live S. pneumoniae organisms derivedfrom the master seed lot by growing the organisms and maintainingthem in aliquots in the freeze-dried form or the frozen state ator below -45 °C. The working seed lot is used, when applicable,after a fixed number of passages, for the inoculation of productionmedium.

Single harvest. The material obtained from one batch of cultures thathave been inoculated with the working seed lot (or with the inoculumderived from it), harvested and processed together.

Purified polysaccharide. The material obtained after final purification.The lot of purified polysaccharide may be derived from a singleharvest or a pool of single harvests processed together.

Modified polysaccharide. Purified polysaccharide that has been mo-dified by chemical reaction or physical process in preparation forconjugation to the carrier.

Carrier. The protein to which the polysaccharide is covalently linkedfor the purpose of eliciting a T-cell dependent immune response tothe pneumococcal polysaccharide.

Monovalent bulk conjugate. A conjugate prepared from a single lot orpool of lots of polysaccharide and a single lot or a pool of lots ofprotein. This is the parent material from which the final bulk isprepared.

Final bulk conjugate. The blend of monovalent conjugates present ina single container from which the final containers are filled, eitherdirectly or through one or more intermediate containers derived fromthe initial single container.

Final lot. A number of sealed, final containers that are equivalent withrespect to the risk of contamination during filling and, when it isperformed, freeze-drying. A final lot must therefore have been filledfrom a single container and freeze-dried in one continuous workingsession.

A.2 General manufacturing recommendations

The general manufacturing recommendations contained in goodmanufacturing practices for pharmaceuticals (28) and biologicalproducts (29) should apply to establishments manufacturing pneumo-coccal conjugate vaccines with the addition of the following:

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Details of standard operating procedures for the preparation andtesting of pneumococcal conjugate vaccines adopted by the manufac-turer together with evidence of appropriate validation of each pro-duction step should be submitted for the approval of the nationalregulatory authority. All assay procedures used for quality control ofthe conjugate vaccines and vaccine intermediates must be validated.Proposals for the modification of manufacturing and control methodsshould also be submitted for approval to the national regulatoryauthority.

Streptococcus pneumoniae is a Biological Safety Level (BSL) 2 patho-gen and represents a particular hazard to health through infection bythe respiratory route. The organism should be handled under condi-tions appropriate for this class of pathogen (30). Standard operatingprocedures need to be developed for dealing with emergencies arisingfrom the accidental spillage, leakage or other dissemination of pneu-mococcal organisms. Personnel employed in the production and con-trol facilities should be adequately trained and appropriate protectivemeasures including vaccination with a pneumococcal vaccine licensedfor use in adults should be implemented. Adherence to current goodmanufacturing practices is important to the integrity of the product,to protect workers and to protect the environment.

A.3 Production controlA.3.1 Control of polysaccharideA.3.1.1 Strains of Streptococcus pneumoniae

The strains of S. pneumoniae used for preparing the polysaccharideshould be agreed with the national regulatory authority. Each strainshould have been shown to be capable of producing polysaccharide ofthe appropriate serotype. Each master seed lot should be identified bya record of its history, including the source from which it was obtainedand the tests done to determine the characteristics of the strain.

The cultures may be examined for the following characteristics: micro-scopically, stained smears from a culture should appear typical of S.pneumoniae; the organism should grow at 37 °C , but not at 25°C, andshould have characteristic smooth alpha haemolytic colonies; the organismshould have the ability to ferment insulin; the organism should be lysed inthe bile solubility test and be sensitive to optochin; a suspension of theculture should be agglutinated or give a positive Quellung reaction with theappropriate serotyping serum.

Nuclear magnetic resonance spectrometry (either 1H or 13C) is a suitablemethod for the confirmation of identity of purified polysaccharide.

A.3.1.2 Seed lot systemThe production of pneumococcal polysaccharide should be based ona working seed lot system. Cultures derived from the working seed

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lots should have the same characteristics as the cultures of the strainfrom which the master seed lot was derived (A.3.1.1). If materials ofanimal origin are used in the medium for seed production, preserva-tion of strain viability for freeze-drying or for frozen storage, thenthey should comply with the guidance given in the Guidelines onTransmissible Spongiform Encephalopathies in Relation to Biologicaland Pharmaceutical Products (31) and should be approved by thenational regulatory authorities.

Manufacturers are encouraged to avoid wherever possible the use ofmaterials of animal origin.

A.3.1.3 Culture media for the production of pneumococcal polysaccharideThe liquid culture medium used for vaccine production should be freefrom ingredients that will form a precipitate upon purification of thecapsular polysaccharide. If materials of animal origin are used thenthey should comply with the guidance given in the Guidelines onTransmissible Spongiform Encephalopathies in Relation to Biologicaland Pharmaceutical Products (31) and should be approved by thenational regulatory authorities.

Manufacturers are encouraged to avoid wherever possible the use ofmaterials of animal origin.

A.3.1.4 Single harvestsConsistency of growth of S. pneumoniae should be demonstrated bymonitoring growth rate, pH and the final yield of polysaccharide.

A.3.1.5 Control of bacterial puritySamples of the culture should be taken before killing and be exam-ined for microbial contamination. The purity of the culture should beverified by suitable methods, which should include inoculation on toappropriate culture media, including plate media that do not supportgrowth of S. pneumoniae. If any contamination is found, the culture orany product derived from it should be discarded. The killing processshould also be adequately validated.

A.3.1.6 Purified polysaccharideEach lot of pneumococcal polysaccharide should be tested for iden-tity, purity and molecular size. A number of approaches to deter-mining polysaccharide identity and purity give complementary butincomplete information, so a combination of methods should be em-ployed to provide all necessary data and should be agreed by thenational regulatory authority. The purity limits given below are ex-pressed with reference to the polysaccharide in its salt form (sodiumor calcium), corrected for moisture. Variations in these specifications

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that may be appropriate if unusual salt forms are present should beagreed by the national regulatory authority.

Generally, after killing the organism, the culture is harvested and thepolysaccharide isolated and purified by techniques such as fractionalprecipitation, chromatography, enzyme treatment and ultrafiltration. Thepolysaccharide is partially purified by fractional precipitation, washed,and dried to a residual moisture content shown to favour the stability ofthe polysaccharide. Methods used for the purification of bulk polysac-charide should be approved by the national regulatory authority. Purifiedpneumococcal polysaccharide and, when necessary, partially purifiedintermediates, are usually stored at or below -20 °C to ensure stability.

A.3.1.6.1 Polysaccharide identity

A test should be performed on the purified polysaccharide to verify itsidentity. In cases where other polysaccharides are produced at thesame manufacturing site, the method should be validated to show thatit distinguishes the desired polysaccharide from all other polysaccha-rides produced at that manufacturing site.

A serological method such as countercurrent immunoelectrophoresis and/or nuclear magnetic resonance spectrometry (either 1H or 13C) is convenientfor this purpose (32–34). In some cases the identity of the polysaccharidecan be deduced from its composition if appropriate analytical methods areemployed.

A.3.1.6.2 Polysaccharide composition

The composition of the polysaccharide provides information on itspurity, identity and the amount of specific impurities, such as pneu-mococcal C-polysaccharide, that are present. Analyses should bebased on the dry weight of the polysaccharide. The composition of thepolysaccharide can be defined in a number of ways depending on themethodology employed and the salt form present. The specificationsused should be agreed by the national regulatory authority.

Chemically, the composition of pneumococcal polysaccharides can bedefined by the percentage of total nitrogen, phosphorus, uronic acid,hexosamine, methyl pentose and O-acetyl groups. These are usuallydetermined by a combination of simple wet chemical tests with colorimetricread outs. Typical specifications are listed in Table 1 (35).

Other methods, such as high performance anion-exchange chroma-tography (HPAEC) with pulsed amperometric detection (HPAEC–PAD)applied to hydrolysates of the polysaccharide, may be used todefineaspects of the quantitative composition of certain polysaccharidetypes, but the method should be validated for the purpose (36). 1H nuclearmagnetic resonance spectrometry also provides a convenient approach forquantitation of the composition of the purified polysaccharide if an internalreference compound is included (33, 34). The proportion of pneumococcalC polysaccharide may be determined by a combination of 1H and31P nuclear magnetic resonance spectrometry (37, 38) or HPAEC–PAD (39).

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A.3.1.6.3 Moisture content

If the purified polysaccharide is to be stored as a lyophilized powder,the moisture content should be determined by suitable methods ap-proved by the national regulatory authority and shown to be withinagreed limits.

A.3.1.6.4 Protein impurity

The protein content should be determined by the method of Lowry etal., using bovine serum albumin as a reference (1, 40), or anothersuitable validated method. Sufficient polysaccharide should be as-sayed to detect 1% protein contamination accurately.

Each lot of purified polysaccharide should typically contain not more than3% by weight of protein. However, this will vary depending upon theserotype and an acceptable level of protein contamination should beagreed with the national regulatory authority.

A.3.1.6.5 Nucleic acid impurity

Each lot of polysaccharide should contain not more than 2% byweight of nucleic acid as determined by ultraviolet spectrophotom-etry, on the assumption that the absorbance of a 1 g/l nucleic acidsolution contained in a cell of 1cm path length at 260 nm is 20 (1) orby another validated method.

Sufficient polysaccharide should be assayed to detect 2% nucleic acidcontamination accurately.

A.3.1.6.6 Pyrogen content

The pyrogen content of the purified polysaccharide should be deter-mined and shown to be within acceptable limits agreed by the na-tional regulatory authority.

A recognized pyrogenicity test can be performed in rabbits. Alternatively,the Limulus amoebocyte lysate test can be performed.

A.3.1.6.7 Molecular size distribution

The molecular size of the purified polysaccharide in each lot providesan indication of the manufacturing consistency. An acceptable levelof consistency should be agreed with the national regulatory authorityand can be established either by process validation or measurementon each lot.

The distribution constant (KD) can be determined by measuring themolecular size distribution of the polysaccharide at the main peak of theelution curve obtained by a suitable chromatographic method. The KD valueand/or the mass distribution limits should be established.

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Methods such as gel filtration through Sepharose CL-4B or CL-6B (orsimilar) in a 0.2 molar buffer using either a refractive index detector orcolorimetric assay for the detection of the polysaccharide; and highperformance size-exclusion chromatography (HPSEC) with refractive indexdetectors either alone or in combination with light scattering (e.g. multipleangle laser light scattering (MALLS)) are suitable for this purpose (34, 41).The methodology and column used should be validated to demonstratesufficient resolution in the appropriate molecular weight range.

A.3.1.7 Modified polysaccharideModified polysaccharide preparations may be partially depolymer-ized either before or during the chemical modification. The registeredpneumococcal conjugate vaccines and several of the candidate vac-cines use polysaccharides and oligosaccharide chains.

A.3.1.7.1 Chemical modification

Several methods are available for the chemical modification ofpolysaccharides before conjugation. The chosen method should beapproved by the national regulatory authority.

The current methods used are similar to those employed in the productionof conjugate vaccines against Haemophilus influenzae type b. For example,polysaccharide may be oxidized with periodate and the periodate-activated polysaccharide attached to free amino groups on the carrierprotein by reductive amination. Alternatively, the polysaccharide canbe randomly activated by cyanogen bromide, or a chemically similarreagent, and a bifunctional linker added, which then allows thepolysaccharide to be attached to the carrier protein directly, or through asecondary linker.

A.3.1.7.2 Extent of modification of the polysaccharide

The manufacturer should demonstrate consistency of the degree ofmodification of the polysaccharide, either by an assay of each batch ofthe polysaccharide or by validation of the manufacturing process.

A.3.1.7.3 Molecular size distribution

The degree of size reduction of the polysaccharide will depend uponthe manufacturing process. The average size distribution (degree ofpolymerization) of the modified polysaccharide should be determinedby a suitable method and shown to be consistent. The molecular sizedistribution should be specified for each serotype, with appropriatelimits for consistency, as the size may affect the reproducibility of theconjugation process.

The molecular size may be determined by gel filtration on soft columns or byHPSEC using refractive index alone, or in combination with laser lightscattering (e.g. MALLS) (34, 41).

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A.3.2 Control of the carrier proteinA.3.2.1 Microorganisms and culture media for production of carrier protein

Microorganisms to be used for the production of the carrier proteinshould be grown in media free from substances likely to cause toxic orallergic reactions in humans. If any materials of animal origin areused in seed preparation or preservation or in production, theyshould comply with the guidance given in the Guidelines on Transmis-sible Spongiform Encephalopathies in Relation to Biological andPharmaceutical Products (31) and should be approved by the nationalregulatory authority.

Production should be based on a seed lot system with the strainsidentified by a record of their history and of all tests made periodicallyto verify strain characteristics. Consistency of growth of the microor-ganisms used should be demonstrated by monitoring the growth rate,pH and final yield of appropriate protein(s).

A.3.2.2 Characterization and purity of the carrier proteinPotentially there are many proteins that could be used as carriers inpneumococcal conjugate vaccines. The principal characteristics of thecarrier protein should be that it is safe and, in the conjugate, elicits aT-cell dependent immune response against the polysaccharide. Testmethods used to characterize such proteins, to ensure that they arenon-toxic and to determine their purity and concentration, should beapproved by the national regulatory authority.

Proteins and purification methods that might be used include:

1. Tetanus or diptheria toxoid. This must satisfy the relevant require-ments p ublished by WHO (42) and be of high purity (43).

2. Diphtheria CRM 197 protein. This is a non-toxic mutant of diph-theria toxin, isolated from cultures of Corynebacterium diphtheriaeC7/b197 (44). Protein purity should be greater than 90% as deter-mined by an appropriate method. When produced in the samefacility as diphtheria toxin, methods must be in place to distinguishthe CRM 197 protein from the active toxin.

The protein carrier should also be characterized. The identity may bedetermined serologically. Physicochemical methods that may be used tocharacterize protein include sodium dodecylsulfate–polyacrylamide gelelectrophoresis (SDS–PAGE), isoelectric focusing, high-performance liquidchromatography (HPLC), amino acid analysis, amino acid sequencing,circular dichroism, fluorescence spectrophotometry (fluorimetry), peptidemapping and mass spectrometry as appropriate (34).

A.3.3 Control of monovalent bulk conjugates

There are a number of possible conjugation methods that might beused for vaccine manufacture; all involve multi-step processes. Both

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the method and the control procedures used to ensure the reproduc-ibility, stability and safety of the conjugate should be established forlicensing. The derivatization and conjugation process should be moni-tored by analysis for unique reaction products or by other suitablemeans. The conditions used in the conjugation process may affect thestructure of the polysaccharide chain by causing the loss of labilesubstituents. Unless the combination of tests used to characterize thebulk monovalent conjugates confirm that the structure is maintained,a test to demonstrate identity of the intact polysaccharide should beperformed.

Residual activated functional groups potentially capable of reactingin vivo may be present following the conjugation process. The manu-facturing process should be validated to show that the activated func-tional groups do not remain at the conclusion of the manufacturingprocess and any residual groups are below a limit approved by thenational regulatory authority.

After the conjugate has been purified, the tests described belowshould be performed in order to assess consistency of manufacture.The tests are critical for assuring lot-to-lot consistency.

A.3.3.1 IdentityA test should be performed on the monovalent bulk to verify itsidentity. The method should be validated to show that it distinguishesthe desired monovalent material from all other polysaccharides andconjugates produced at that manufacturing site.

A.3.3.2 Residual reagentsThe conjugate purification procedures should remove residualreagents used for conjugation and capping. The removal of rea-gents and reaction by-products such as cyanide, 1-ethyl-3,3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and others, dependingon the conjugation chemistry, should be confirmed by suitable tests orby validation of the purification process.

A.3.3.3 Polysaccharide–protein ratio and conjugation markersFor each batch of the bulk conjugate of each serotype, the ratio ofpolysaccharide to carrier protein should be determined as a marker ofthe consistency of the conjugation chemistry. For each conjugate, theratio should be within the range approved for that particular conju-gate by the national regulatory laboratory and should be consistentwith vaccine shown to be effective in clinical trials.

For pneumococcal conjugate vaccines the ratio is typically in the range of0.3–3.0 but varies with the serotype. The ratio can be determined either byindependent measurement of the amounts of protein and polysaccharide

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present (corrected for unbound protein and unbound polysaccharide), orby methods that give a direct measure of the ratio. Methods include 1Hnuclear magnetic resonance spectroscopy or the use of HPSEC with dualmonitoring (e.g. refractive index and ultraviolet, for total material and proteincontent, respectively).

If the chemistry of conjugation results in the creation of a uniquelinkage marker (e.g. a unique amino acid), each batch of the bulkconjugate of that serotype should be assessed to quantify the degreeof substitution of the carrier protein by covalent reaction with themodified pneumococcal polysaccharide.

The structural complexity and structural differences between thepneumococcal serotypes are such that in most cases a simple conjugationmarker will not be able to be identified.

A.3.3.4 Capping markersEach batch should be shown to be free of activated functional groupson either the chemically modified polysaccharide or carrier protein.Alternatively, the product of the capping reaction can be monitored,or the capping reaction can be validated to show removal ofunreacted functional groups. Validation of the manufacturing processduring vaccine development can eliminate the need to perform thisanalysis for routine control.

A.3.3.5 Conjugated and unbound (free) polysaccharideOnly the pneumococcal polysaccharide that is covalently bound tothe carrier protein, i.e. conjugated polysaccharide, is immunologicallyimportant for clinical protection. Each batch of conjugate should betested for unbound or free polysaccharide in order to ensure that theamount present in the purified bulk is within the limits agreed by thenational regulatory authority based on lots shown to be clinically safeand efficacious.

Methods that have been used to separate unbound polysaccharide prior toassay, that are potentially applicable to pneumococcal conjugates, includehydrophobic chromatography, acid precipitation, precipitation with carrier-protein-specific antibodies, gel filtration and ultrafiltration. The amountof unbound polysaccharide can be determined by specific chemical orimmunological tests, or by HPAEC after hydrolysis.

A.3.3.6 Protein contentThe protein content of the conjugate should be determined by meansof an appropriate validated assay and should comply with limits forthe particular product. Each batch should be tested for conjugatedand unbound protein.

If possible, the unconjugated protein should also be measured. Appropriatemethods for the determination of conjugated and unconjugated proteininclude HPLC or capillary electrophoresis.

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A.3.3.7 Molecular size distribution of polysaccharide–protein conjugateThe molecular size of the polysaccharide–protein conjugate is animportant parameter in establishing consistency of production and instudying stability during storage.

The relative molecular size of the polysaccharide–protein conjugateshould be determined for each bulk using a gel matrix appropriate tothe size of the conjugate. The method should be validated with anemphasis on having sufficient specificity to distinguish the polysaccha-ride–protein conjugate from other components that may be pre-sent, e.g. unbound protein or polysaccharide. The size-distributionspecifications will be vaccine-specific and should be consistent withlots shown to be immunogenic in clinical trials.

Typically the size may be determined by gel filtration on Sepharose CL-2B,or by HPSEC on an appropriate column. Because the polysaccharide–protein ratio is an average value, characterization of this ratio over theconjugates with their size distribution (e.g. by dual monitoring of the columneluent) can be used to provide further proof of manufacturing consistency(46).

A.3.3.8 SterilityThe bulk purified conjugate should be tested for bacterial and mycoticsterility in accordance with the recommendations of Part A, sections5.1 and 5.2, of the revised Requirements for the Sterility of BiologicalSubstances (47) or by a method approved by the national regulatoryauthority. If a preservative has been added to the product, appropri-ate measures should be taken to prevent it from interfering with thetest.

A.3.3.9 Specific toxicity of carrier proteinThe bulk conjugate should be tested for the absence of specific toxic-ity of the carrier protein where appropriate (e.g. when tetanus ordiphtheria toxoids have been used). Absence of specific toxicity of thecarrier protein may also be assessed through validation of the produc-tion process.

A.3.3.10Endotoxin contentTo ensure an acceptable level of endotoxin in the final product, theendotoxin content of the monovalent bulk may be determined andshown to be within acceptable limits agreed by the national regulatoryauthority.

A.3.4 Final bulkA.3.4.1Preparation

To formulate the final bulk, monovalent conjugate bulks may bemixed together and an adjuvant, a preservative and/or stabilizer is

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added before final dilution. Alternatively, the monovalent conjugatebulks may be adsorbed to adjuvant individually before mixing them toformulate the final vaccine.

A.3.4.2SterilityEach final bulk should be tested for bacterial and mycotic sterility asindicated in section. A.3.3.7.

A.3.5 Filling and containers

The recommendations concerning filling and containers given in An-nex 1, Section 4 of Good Manufacturing Practices for BiologicalProducts (39) should be applied (29).

A.3.6 Control tests on final productA.3.6.1 Identity

An identity test should be performed that demonstrates that all of theintended pneumococcal polysaccharide serotypes are present in thefinal product, unless this test has been performed on the final bulk.

A serological test, using antibodies specific for the purified polysaccharidemay be used.

A.3.6.2 SterilityThe contents of final containers should be tested for bacterial andmycotic sterility as indicated in section A.3.3.8.

A.3.6.3 Pneumococcal polysaccharide contentThe amount of each pneumococcal polysaccharide in the final con-tainers should be determined, and shown to be within the specifica-tions agreed by the national regulatory authority.

The conjugate vaccines produced by different manufacturers differ informulation. A quantitative assay for each of the pneumococcalpolysaccharides in the final container should be carried out. The assaysused are likely to be product-specific and might include chromatographicor serological methods. Immunological assays such as rate nephelometry(48) or ELISA inhibition may be used.

A.3.6.4 Residual moistureIf the vaccine is freeze-dried, the average moisture content should bedetermined by methods accepted by the national regulatory author-ity. Values should be within limits for the preparations shown to beadequately stable in the stability studies of the vaccine.

The test should be performed on 1 vial per 1000 up to a maximum of 10 vialsbut on no less than 5 vials taken at random from throughout the final lot. Theaverage residual moisture content should generally be no greater than2.5% and no vial should be found to have a residual moisture content of 3%or greater.

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A.3.6.5 Endotoxin contentThe vaccine in the final container should be tested for endotoxincontent by a Limulus amoebocyte lysate (LAL) test. Endotoxin con-tent or pyrogenic activity should be consistent with levels found to beacceptable in vaccine lots used in clinical trials and approved by thenational regulatory authority.

A.3.6.6 Adjuvant contentIf an adjuvant has been added to the vaccine, its content should bedetermined by a method approved by the national regulatory author-ity. The amount and nature of the adjuvant should be agreed with thenational regulatory authority. If aluminium compounds are used asadjuvants, the amount of aluminium should not exceed 1.25mg persingle human dose.

A.3.6.7 Preservative contentThe manufacturer has a choice of possible preservatives. Consider-ation should be given to the stability of the chosen preservative andpossible interactions between the vaccine components and the preser-vative. If a preservative has been added to the vaccine, the content ofpreservative should be determined by a method approved by thenational regulatory authority. The amount of preservative in the vac-cine dose should be shown not to have any deleterious effect on theantigen or to impair the safety of the product in humans. The preser-vative and its concentration should be approved by the national regu-latory authority.

A.3.6.8 General safety test (innocuity)The requirement to test lots of pneumococcal conjugate vaccine forunexpected toxicity (abnormal toxicity) should be agreed with thenational regulatory authority.

Such a test may be omitted for routine lot release once consistencyof production has been well established to the satisfaction of thenational regulatory authority and when good manufacturing practice is inplace.

A.3.6.9 pHIf the vaccine is a liquid preparation, the pH of each final lot should betested and shown to be within the range of values found for vaccinelots shown to be safe and effective in the clinical trials and in stabilitystudies. For a lyophilized preparation, the pH should be measuredafter reconstitution with the appropriate diluent.

A.3.6.10 Inspection of final containersEach container in each final lot should be inspected visually (manu-ally or with automatic inspection systems), and those showing abnor-

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malities, lack of integrity and, if applicable, clumping or the presenceof particles should be discarded.

A.4 Records

The recommendations in section 8 of Good manufacturing practicesfor biological products (39, Annex 1) should be applied (29).

A.5 Retained samples

The recommendations in section 9.5 of Good manufacturing practicesfor biological products (39, Annex 1) should be applied (29).

A.6 Labelling

The recommendations in section 7 of Good manufacturing practicesfor biological products (39, Annex 1) should be applied with theaddition of the following (29).

The label on the carton or the leaflet accompanying the containershould indicate:

— the pneumococcal serotype and carrier protein present in eachsingle human dose;

— the amount of each conjugate present in a single human dose;— the temperature recommended during storage and transport;— if the vaccine is freeze-dried, that after its reconstitution it

should be used immediately unless data have been provided tothe licensing authority that it may be stored for a limited time;and

— the volume and nature of the diluent to be added in order toreconstitute a freeze-dried vaccine, specifying that the diluentshould be supplied by the manufacturer and approved by thenational regulatory authority.

A.7 Distribution and transport

The recommendations in section 8 of Good manufacturing practicesfor biological products (39, Annex 1) should be applied (29).

A.8 Stability, storage and expiry dateA.8.1 Stability testing

Adequate stability studies form an essential part of the vaccine devel-opment process. The stability of the vaccine in its final form and at therecommended storage temperatures should be demonstrated to thesatisfaction of the national regulatory authority with final containersfrom at least three lots of final product made from different indepen-dent bulk conjugates.

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Given the complexity of these multivalent vaccines, other approaches maybe used, with the approval of the national regulatory authority.

The polysaccharide component of conjugate vaccines may be subjectto gradual hydrolysis at a rate that may vary depending upon the typeof conjugate, the type of formulation or adjuvant, the types of excipi-ent and conditions of storage. The hydrolysis may result in reducedmolecular size of the pneumococcal polysaccharide component, areduction in the amount of the polysaccharide bound to the proteincarrier and in a reduced molecular size of the conjugate.

The structural stability of the oligosaccharide chains and of the proteincarrier vary between different conjugate vaccines.

Tests should be conducted before licensing to determine the extent towhich the stability of the product has been maintained throughout theproposed validity period. The vaccine should meet the specificationsfor final product up to the expiry date.

Molecular sizing of the final product may be carried out to ensure theintegrity of the conjugate. The antigen content of each serotype conjugatemay be determined by a quantitative serological assay.

The desorption of antigen from aluminium-based adjuvants, ifused, may take place over time. The level of adsorption should beshown to be within limits agreed by the national regulatory authority,unless data are available to show that the immunogenicity of thefinal product is not dependent upon adsorption of the antigen to theadjuvant.

Accelerated stability studies may provide additional supporting evi-dence of the stability of the product but cannot replace real-timestudies.

When any changes are made in the production procedure that mayaffect the stability of the product, the vaccine produced by the newmethod should be shown to be stable.

The statements concerning storage temperature and expiry dateappearing on the label should be based on experimental evidence,which should be submitted for approval to the national regulatoryauthority.

A.8.2 Storage conditions

Storage conditions should be based on stability studies and approvedby the national regulatory authority.

Storage of both liquid and freeze-dried vaccines at a temperature of 2–8°Chas been found to be satisfactory. The stability of pneumococcal conjugatecomponents varies with serotype of the capsular polysaccharide.

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A.8.3 Expiry date

The expiry date should be approved by the national regulatory au-thority and based on the stability of the final product as well as theresults of the stability tests referred to in section A.8.1.

Part B. Requirements for national regulatoryauthorities

B.1 General

The general recommendations for control laboratories contained inthe Guidelines for National Authorities on Quality Assurance forBiological Products (29) should be applied.

B.2 Official release and certification

A vaccine lot should be released only if it fulfils national requirementsand/or Part A of these Recommendations.

A statement signed by the appropriate official of the national regula-tory authority should be provided at the request of the manufacturingestablishments and should certify that the lot of vaccine in questionsatisfies all national requirements as well as Part A of these Recom-mendations. The certificate should state the number under which thelot was released by the national controller, and the number appearingon the labels of the containers. Importers of pneumococcal conjugatevaccines should be given a copy of the official national release docu-ment. The purpose of the certificates is to facilitate the exchange ofvaccines between countries.

B.3 Reactogenicity and immunogenicity of vaccine in humans

The national regulatory authority should satisfy itself that adequatecontrol of the pneumococcal conjugate vaccine has been achieved.Clinical data supporting consistency of vaccine production should beobtained prior to registration of the product. Several different lots ofthe product should be used during the clinical studies and shown togive similar immune responses. Such studies may need to be repeatedif changes in production are made, or when the pneumococcal conju-gate is intended to be part of a new combination vaccine formulation.The national regulatory authority should ensure that the studies in-clude an adequate number of subjects to provide statistically validdata on reactivity and immunogenicity. The pneumococcal conjugatevaccines are manufactured from purified components by a clearlydefined chemical process. Any changes in production or formulationof the vaccine should be reported to the national regulatory authority,

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which will decide whether additional clinical data are required on acase-by-case basis. Such a review should take into account the likeli-hood of such changes affecting the quality, the consistency, the struc-tural integrity and the immunogenicity of the product, and considerthe possible cumulative effect of multiple modifications that individu-ally may be regarded as minor.

AuthorsThe first draft of these Recommendations was prepared by Dr I. Feavers, Divisionof Bacteriology, National Institute for Biological Standards and Control, Potters Bar,Herts., England; Dr C. Frasch, Laboratory for Bacterial Polysaccharides, Center forBiologics Evaluation and Research, Food and Drug Administration, Rockville, MD,USA; Dr C. Jones, Laboratory of Molecular Structure, National Institute for Biologi-cal Standards and Control, Potters Bar, Herts., England; Dr N. Ravenscroft, De-partment of Chemistry, University of Cape Town, Rondebosch, South Africa.

The second draft was prepared after a WHO Informal Consultation held in Geneva,4–5 June 2003 attended by the following participants:

Dr G. Carlone, Respiratory Diseases Branch, Division of Bacterial and MycoticDisease, National Center for Infectious Diseases, Atlanta, GA, USA; Dr N.Cauwenberghs, Regulatory Affairs Paediatric Vaccines, GlaxoSmithKlineBiologicals, Rixensart, Belgium; Dr C. Ceccarini, Siena, Italy; Dr E.C. Leal,Fundaçao Oswaldo Cruz, National Institute for Quality Control, Rio de Janeiro,Brazil; Dr R. Dagan, Soroka Medical Center, Paediatric Infectious Disease Unit,Beer Sheva, Israel; Dr J. Eskola, Aventis Pasteur, Lyon, France; Dr I. Feavers,Division of Bacteriology, National Institute for Biological Standards and Control,Potters Bar, Herts., Dr C. Frasch, Laboratory for Bacterial Polysaccharides, Centerfor Biologics Evaluation and Research, Food and Drug Administration, Rockville,MD, USA; Dr D. Goldblatt, Institute of Child Health, Microbiology Unit, UniversityCollege London Medical School, London, England; Dr E. Griffiths, Biologics andGenetic Therapies Directorate, Tunney’s Pasture, Ottawa, Canada; Dr C. Jones,Laboratory of Molecular Structure, National Institute for Biological Standards andControl, Potters Bar, Herts., England; Dr M.H. Käyhty, Department of Vaccines,National Public Health Institute, Helsinki, Finland; Professor K. Klugman, Depart-ment of International Health, The Rollins School of Public Health, Emory University,Atlanta, GA, USA; Dr R.C. Kohberger, Statistics and Data Management, Wyeth-Lederle Vaccines, Pearl River, New York, USA; Dr O. Levine, Bloomberg Schoolof Public Health, Department of International Health, Johns HopkinsUniversity, Baltimore, MD, USA; Dr P. Lommel, Research and Development,GlaxoSmithKline Biologicals, Rixensart, Belgium; Dr J. Maleckar, Aventis Pasteur,Swiftwater, PA, USA; Dr M. Nahm, Department of Pathology, University of Alabamaat Birmingham, Birmingham, AL, USA; Dr K. O’Brien, Johns Hopkins School ofHygiene and Public Health, Center for American Indian Health, Baltimore, MD,USA; Dr V. Öppling, Paul Ehrlich Institute, Langen, Germany; Dr B. Plikaytis,Division of Bacterial and Mycotic Diseases, National Center for Infectious Dis-eases, Centers for Disease Control and Prevention, Atlanta, GA, USA; Dr J.Poolman, Research and Development, GlaxoSmithKline Biologicals, Rixensart,Belgium; Dr D.R. Pratt, Division of Vaccines and Related Product Applications, USFood and Drug Administration, Rockville MD, USA; Dr N. Ravenscroft, Department

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of Chemistry, University of Cape Town, Cape Town, South Africa; Dr G.R. Siber,Wyeth Lederle Vaccines and Pediatrics, Pearl River, NY, USA; Dr Sook-JinHur, Division of Bacterial Products, Department for Biologics Evaluation,Korea Food and Drug Administration, Seoul, Republic of Korea; Dr B. Thirion,GlaxoSmithKline Biologicals, Rixensart, Belgium; Dr N. Tornieporth, Researchand Development, GlaxoSmithKline Biologicals, Rixensart, Belgium; Dr I. Uhnoo,Medical Product Agency, Uppsala, Sweden; Dr A.R.T. Utami, National Agencyof Drug and Food Control Indonesia, Jakarta, Indonesia; Dr C. Whitney, Respira-tory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Centerfor Infectious Diseases, Centers for Disease Control and Prevention, Atlanta,GA, USA.

WHO Secretariat: Dr T. Cherian, Initiative for Vaccine Research, Vaccines andBiologicals, World Health Organization, Geneva, Switzerland; Dr Hong-ki Min,Quality Assurance and Safety of Biologicals, Vaccines and Biologicals, WorldHealth Organization, Geneva, Switzerland; Dr D. Wood, Quality Assurance andSafety of Biologicals, Vaccines and Biologicals, World Health Organization,Geneva, Switzerland.

Acknowledgements are due to the following experts for their useful comments onthe second draft:

Dr N. Cauwenberghs, Regulatory Affairs Paediatric Vaccines, GlaxoSmithKlineBiologicals, Rixensart, Belgium; Dr M. Nahm, Department of Pathology, Universityof Alabama at Birmingham, Birmingham, AL, USA; Dr A.R.T. Utami, NationalAgency of Drug and Food Control, National Agency of Drug and Food ControlIndonesia, Jakarta, Indonesia.

References1. Requirements for meningococcal polysaccharide vaccine. In: WHO Expert

Committee on Biological Standardization. Twenty-fifth report. Geneva,World Health Organization, 1981 (WHO Technical Report Series, No. 658),p. 29.

2. Jedrzejas MJ. Pneumococcal virulence factors: structure and function.Microbiology and Molecular Biology Reviews, 2001, 65:187–207.

3. Robbins JB et al. The 1996 Albert Laske Medical Research Awards.Prevention of systemic infections, especially meningitis, caused byHaemophilus influenzae type b. Impact on public health and implications forother polysaccharide-based vaccines. Journal of the American MedicalAssociation, 1996, 276:1181–1185.

4. Gray BM, Converse GM, III, Dillon HC, Jr. Epidemiologic studies ofStreptococcus pneumoniae in infants: acquisition, carriage, and infectionduring the first 24 months of life. Journal of Infectious Diseases, 1980,142:923–933.

5. Musher DM. Infections caused by Streptococcus pneumoniae: clinicalspectrum, pathogenesis, immunity, and treatment. Clinical InfectiousDiseases, 1992, 14:801–807.

6. Henrichsen J. Six newly recognized types of Streptococcus pneumoniae.Journal of Clinical Microbiology, 1995, 33:2759–2762.

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7. Eskola J, Anttila M. Pneumococcal conjugate vaccines. Pediatric InfectiousDisease Journal, 1999, 18:543–551.

8. Black SB et al. Postlicensure evaluation of the effectiveness of seven valentpneumococcal conjugate vaccine. Pediatric Infectious Disease Journal,2001, 20:1105–1107.

9. Wuorimaa T et al. Avidity and subclasses of IgG after immunization ofinfants with an 11-valent pneumococcal conjugate vaccine with or withoutaluminum adjuvant. Journal of Infectious Diseases, 2001, 184:1211–1215.

10. Huebner RE et al. Immunogenicity after one, two or three doses and impacton the antibody response to coadministered antigens of a nonavalentpneumococcal conjugate vaccine in infants of Soweto, South Africa.Pediatric Infectious Disease Journal, 2002, 21:1004–1007.

11. Rennels MB et al. Safety and immunogenicity of heptavalent pneumococcalvaccine conjugated to CRM197 in United States infants. Pediatrics, 1998,101:604–611.

12. Shinefield HR et al. Safety and immunogenicity of heptavalentpneumococcal CRM197 conjugate vaccine in infants and toddlers. PediatricInfectious Disease Journal, 1999, 18:757–763.

13. Black S et al. Efficacy, safety and immunogenicity of heptavalentpneumococcal conjugate vaccine in children. Northern California KaiserPermanente Vaccine Study Center Group. Pediatric Infectious DiseaseJournal, 2000, 19:187–195.

14. Eskola J. Immunogenicity of pneumococcal conjugate vaccines. PediatricInfectious Disease Journal, 2000, 19:388–393.

15. Hausdorff WP et al. Which pneumococcal serogroups cause the mostinvasive disease: implications for conjugate vaccine formulation and use,part I. Clinical Infectious Diseases, 2000, 30:100–121.

16. Nurkka A et al. Serum and salivary anti-capsular antibodies in infants andchildren vaccinated with octavalent pneumococcal conjugate vaccines,PncD and PncT. Vaccine, 2001, 20:194–201.

17. Klugman KP et al. A trial of a 9-valent pneumococcal conjugate vaccine inchildren with and those without HIV infection. New England Journal ofMedicine, 2003, 349:1341–1348.

18. Mbelle N et al. Immunogenicity and impact on nasopharyngeal carriage of anonavalent pneumococcal conjugate vaccine. Journal of InfectiousDiseases, 1999, 180:1171–1176.

19. Pelton SI et al. Pneumococcal conjugate vaccines: proceedings from aninteractive symposium at the 41st Interscience Conference on AntimicrobialAgents and Chemotherapy. Vaccine, 2003, 21:1562–1571.

20. Schiffman G et al. A radioimmunoassay for immunologic phenomena inpneumococcal disease and for the antibody response to pneumococcalvaccines. I. Method for the radioimmunoassay of anticapsular antibodiesand comparison with other techniques. Journal of Immunological Methods,1980, 33:133–144.

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21. Nahm MH, Siber GR, Olander JV. A modified Farr assay is more specificthan ELISA for measuring antibodies to Streptococcus pneumoniae capsularpolysaccharides. Journal of Infectious Diseases, 1996, 173:113–118.

22. Quataert SA et al. Assignment of weight-based antibody units to a humanantipneumococcal standard reference serum, lot 89-S. Clinical andDiagnostic Laboratory Immunology, 1995, 2:590–597.

22a.Wernette CM, Frasch CE, Madore D et al. Enzyme-linked immunosorbentassay for quantitation of human antibodies to pneumococcalpolysaccharides. Clinical and Diagnostic Laboratory Immunology, 2003,10:514–519.

23. Guckian JC, Christensen GD, Fine DP. The role of opsonins in recoveryfrom experimental pneumococcal pneumonia. Journal of InfectiousDiseases, 1980, 142:175–190.

24. Vitharsson G et al. Opsonization and antibodies to capsular and cell wallpolysaccharides of Streptococcus pneumoniae. Journal of InfectiousDiseases, 1994, 170:592–599.

25. Romero-Steiner S et al. Standardization of an opsonophagocytic assay forthe measurement of functional antibody activity against Streptococcuspneumoniae using differentiated HL-60 cells. Clinical and DiagnosticLaboratory Immunology, 1997, 4:415–422.

26. Plikaytis BD et al. An analytical model applied to a multicenterpneumococcal enzyme-linked immunosorbent assay study. Journal ofClinical Microbiology, 2000, 38:2043–2050.

27. Choo S, Finn A. Pediatric combination vaccines. Current Opinion inPediatrics, 1999, 11:14–20.

28. Good manufacturing practices for pharmaceutical products. In: WHO ExpertCommittee on Specifications for Pharmaceutical Preparations. Thirty-secondreport. Geneva, World Health Organization, 1992 (WHO Technical ReportSeries, No. 823).

29. Good manufacturing practices for biological products. In: WHO ExpertCommittee on Biological Standardization. Forty-second report. Geneva,World Health Organization, 1992 (WHO Technical Report Series, No. 822).

30. Biosafety guidelines for personnel engaged in the production of vaccinesand biologicals for medical use. Geneva, World Health Organization, 1995(WHO/CDS/BVI/95.5).

31. Guidelines on transmissible spongiform encephalopathies in relation tobiological and pharmaceutical products, Geneva, World HealthOrganization, 2003 (WHO/BCT/QSD/03.01).

32. Jones C. Capsular polysaccharides from Neisseria meningitidis andStreptococcus pneumoniae. Carbohydrates in Europe, 1998, 21:11–16.

33. Abeygunawardana C et al. Development and validation of an NMR-basedidentity assay for bacterial polysaccharides. Analytical Biochemistry, 2000,279:226–240.

34. Hsieh CL. Characterization of saccharide-CRM197 conjugate vaccines.Developments in Biologicals, 2000, 103:93–104.

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35. Pneumococcal polysaccharide vaccine. In: European Pharmacopoeia, 4thed. Strasbourg, European Directorate for the Quality of Medicine,2002:2205–2206.

36. Talaga P, Vialle S, Moreau M. Development of a high-performance anion-exchange chromatography with pulsed amperometric detection basedquantification assay for pneumococcal polysaccharides and conjugates.Vaccine, 2002, 20:2474–2484.

37. Jones C, Currie F. Control of components of bacterial polysaccharidevaccines by physical methods. Biologicals, 1991, 19:41–47.

38. Wu XD et al. Determination of C-polysaccharide in pneumococcalpolysaccharides using 1H and 31P NMR. Developments in Biologicals,2000, 103:269–270.

39. Talaga P, Bellamy L, Moreau M. Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides byuse of high-performance anion-exchange chromatography with pulsedamperometric detection. Vaccine, 2001, 19:2987–2994.

40. Lowry OH et al. Protein measurement with the Folin phenol reagent. Journalof Biological Chemistry, 1951, 193:265–275.

41. Bednar B, Hennessey JP, Jr. Molecular size analysis of capsularpolysaccharide preparations from Streptococcus pneumoniae.Carbohydrate Research, 1993, 243:115–130.

42. Requirements for diphtheria, tetanus, pertussis and combined vaccines.In: WHO Expert Committee on Biological Standardization. Fortieth report.Geneva, World Health Organization, 1990 (WHO Technical Report SeriesNo. 800).

43. Tetanus vaccine (adsorbed). In: European Pharmacopoeia, 4th ed.Strasbourg, European Directorate for the Quality of Medicines, 2002:2216–2217.

44. Giannini G, Rappuoli R, Ratti G. The amino acid sequence of two non-toxicmutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Research1984, 12:4063–4069.

45. Jones C et al. Spectroscopic studies of the structure and stability ofglycoconjugate vaccines. Developments in Biologicals, 2000, 103:121–136.

46. Ravenscroft N et al. Physicochemical characterization of theoligosaccharide component of vaccines. Developments in Biologicals, 2000,103:35–47.

47. General requirements for the sterility of biological substances. In: WHOExpert Committee on Biological Standardization. Twenty-fifth report. Geneva,World Health Organization, 1973 (WHO Technical Report Series, No. 530).

48. Lee CJ. Quality control of polyvalent pneumococcal polysaccharide-proteinconjugate vaccine by nephelometry. Biologicals, 2002, 30:97–103.

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AppendixSerological criteria for evaluation and licensure ofnew pneumococcal conjugate vaccine formulationsfor use in infants

The lack of a definitive serological correlate of protection and themultiplicity of antigens involved, especially because the clinical effi-cacy of several of the individual serotypes represented in the onlylicensed vaccine has not been established, have been an obstaclefor licensure of new formulations or combinations of pneumococcalconjugate vaccines.

WHO undertook a series of consultations to develop serological cri-teria for the evaluation and licensure of new formulations, combina-tions or different vaccination schedules for pneumococcal conjugatevaccines. At a consultation held in Alaska in May 2002, a preliminaryanalysis of data from the efficacy trial in northern California waspresented. The results of the analysis showed that a threshold anti-body concentration for protection against invasive disease could beestimated using a few simplifying assumptions, and the followingrelationship between the point estimate of clinical efficacy (VE) anda protective antibody concentration:

VE 1

Probability of disease in Vax groupProbability of disease in control group

= -

\ = -VE 1

% of Vax subjects with [Ab] < Ab% of control subjects with [Ab] < Ab

protective

protective

where Vax group is the vaccinated group and [Ab] is concentration ofantibody.

The threshold antibody concentration of 0.20mg/ml thus derived wassupported by a number of other observations. These included:

— the threshold corresponded with the threshold opsonophagocyticantibody titre of 1 : 8;

— it predicted age-specific disease rates;— it was consistent with available data from passive immunization

using bacterial polysaccharide immune globulin (BPIG) toprevent pneumococcal otitis media and invasive pneumococcaldisease;

— it appeared to discriminate clearly between vaccinees who hadreceived conjugate and controls in immunogenicity studies;

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— infants with antibody above the threshold showed evidenceof priming and a booster response to a subsequent dose ofvaccine. The rationale for selecting the threshold antibodyconcentration is described in more detail in the proceedings of aWHO meeting (1).

On the recommendations arising from this consultation, this analysiswas repeated using the pooled immunogenicity and efficacy data fromall the completed trials of pneumococcal conjugate vaccines to nar-row the confidence limits around the point-estimate of efficacy and toallow additional populations to be represented. The threshold anti-body concentration derived from the pooled analysis using the meth-ods described previously was 0.35mg/ml. Opsonophagocytic antibodytitres were available from two of the three studies and analysis of thedata showed that antibody concentrations in the range of 0.20–0.35 mg/ml correlated best with an opsonophagocytic antibody titre of1 :8, which in turn correlates best with protective efficacy. The resultsof the pooled analysis were presented at a second consultation held inJune 2003, which was attended by experts in pneumococcal epidemi-ology and vaccine evaluation, as well as representatives of regulatoryagencies. On the basis of the data presented at this consultation, thecriteria listed in the following section were recommended for use as arelevant value to establish non-inferiority of a new vaccine whencompared to a vaccine against invasive pneumococcal disease thatis already licensed. These criteria should not be used to evaluatevaccines against other clinical end-points, e.g. pneumonia and otitismedia. It should be noted that immunological responses to pneu-mococcal conjugate vaccines may vary significantly by population,and a new candidate vaccine shown to be inferior to the licensedvaccine in one population may nevertheless be non-inferior in asecond population and may therefore be acceptable in the secondpopulation.

The development of standardized assays to evaluate serological re-sponses to new pneumococcal conjugate vaccines has been longpursued by WHO through many consultations. Agreement wasreached at a WHO Workshop held in Geneva in 2000 to select onewell-characterized pneumococcal ELISA protocol as a reference orbenchmark assay for laboratories evaluating serological responses topneumococcal vaccines and to make the link with the pivotal clinicalprotection studies carried out during the licensure of the first seven-component conjugate vaccine. Two WHO reference laboratorieshave been established to help other laboratories set up and standard-ize their own pneumococcal ELISA and to ensure the comparabilityand acceptability of the serological data. These reference laboratories

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are located at the Institute of Child Health, London, England , andat the Bacterial Respiratory Pathogen Reference Laboratory, TheUniversity of Birmingham, Alabama, USA. The detailed protocol forthe pneumococcal ELISA, developed with technical assistance fromWyeth Vaccines, Rochester, New York, USA, is available throughthe Internet site at: www.vaccine.uab.edu.

Primary end-point

The following criteria are recommended for use as the primaryend-point for demonstration of non-inferiority against a registeredvaccine:

• IgG antibody concentration, as measured by ELISA, in seracollected 4 weeks after a three-dose primary series is consideredto be the optimal primary end-point and main licensingparameter.

• A single threshold or reference antibody concentration is re-commended for use for all pneumococcal serotypes. A referenceantibody concentration of 0.35mg/ml, that has been determinedthrough a pooled analysis of data from the efficacy trials withinvasive disease end-points that have been completed to date, isrecommended (1, 2). This threshold does not necessarily predictprotection in an individual subject.

• The reference value is defined on the basis of data obtained usingELISA without pre-adsorption with serotype 22F. Antibody con-centrations determined using an alternative method will need to bebridged to this method to derive an equivalent threshold concentra-tion. It is recommended that the assay used be calibrated against areference assay (3).

• Direct clinical comparison of the registered (established) vaccinewith the new one is the preferred method for evaluating newvaccine formulations.

• The percentage of responders (those in whom post-immunizationantibody concentration is above the threshold) should be used asthe criterion to determine non-inferiority.

• For the serotypes present in a registered vaccine, the percentage ofresponders to each serotype in the new formulation or combinationshould be compared with the percentage of responders to the sameserotype in the registered vaccine in the same population. Non-inferiority to antibody response for each of the serotypes in theregistered vaccine is desirable, but not an absolute requirement.Registration of products in which one or more serotypes do notmeet non-inferiority criteria would have to be decided on an indi-vidual basis.

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• Serotypes not contained in a registered formulation may be evalu-ated for non-inferiority to the aggregate response to the serotypesin the registered vaccine. Failure of one or more new serotypesto meet this criterion may be considered on an individual basis(see example given above).

Additional criteria that must be met to support registration

In addition to showing non-inferiority with respect to the primaryend-point, additional data to demonstrate the functional capacity ofthe antibody and induction of immunological memory in a subset ofthe sera are required for registration.

Functional antibodies

• Opsonophagocytic activity as measured by opsonophagocytic assayafter a three-dose priming series is required to demonstrate thefunctionality of antibodies.

• The method used to demonstrate opsonophagocytic activity shouldbe comparable to the reference assay (4).

Immunological memory

• Evidence of memory should be demonstrated. One possiblemethod is to administer a booster dose of pneumococcal poly-saccharide vaccine and to compare concentrations between age-matched unprimed and primed individuals; data from non-concur-rent controls may be sufficient for the purposes of comparison.

• A full dose of polysaccharide vaccine should be used at this stagebecause the use of a reduced dose of the polysaccharide vaccine asa booster has not been sufficiently tested.

• Avidity of antibodies is also a useful marker for immunologicalmemory.

The following reference reagents and quality control and referencematerials are available for the serological assays (also available at:http://www.vaccine.uab.edu/information.htm) (see also Tables A1,A2a and A2b).

Pneumococcal ELISA calibration sera: To obtain an aliquot of eachof the 12 sera please contact (email preferred):

Dr David GoldblattEmail: d.goldblatt@ich.ucl.ac.ukWHO Pneumococcal Reference LaboratoryInstitute of Child Health

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Table A.2aValues assigned to 89-SF (reference serum)

Typea Total antibody IgG (mg/l)b IgM (mg/l) IgA (mg/l)(mg/l)

1 10.7 6.3 1.7 1.43 7.9 2.4 0.6 4.34 7.0 4.1 1.4 1.25 10.0 5.8 4.2 1.26B 24.3 16.9 3.0 1.57F 7.3 5.2 1.9 1.19V 10.2 6.9 1.6 1.714 37 27.8 1.2 1.918C 6.7 4.5 1.3 0.819F 18.8 13.0 3.2 2.0223F 11.9 8.1 0.7 1.3

a Serotypes 1 and 5 are included in a typical 9-valent vaccine and serotypes 3 and 7F areincluded in a typical 11-valent vaccine.

b Source: reference 5, confirmed by CBER, USFDA. The value assigned for 19F is subject tofurther confirmation.

Table A.2bValues assigned to 89-SF for additional serotypes

Type Total Ig (mg/l) IgG (mg/l) IgM (mg/l) IgA (mg/l)

2 21.4 12.2 5.1 3.98 11.5 5.1 2.0 2.09N 12.7 7.8 2.4 2.1

30 Guilford StreetLondon WC1N 1EH, England

Sera are stored at, and will be distributed by, the National Institute ofBiological Standards and Control, Potters Bar, Herts., England.

References1. Jodar L et al. Serological criteria for evaluation and licensure of new

pneumococcal conjugate vaccine formulations for use in infants. Vaccine,2003, 21:3265–3272.

2. Chang I et al. Serological predictors against invasive pneumococcal disease:a summary of three pneumococcal conjugate vaccine efficacy trials. Paperpresented to Consultation Meeting on WHO Guidelines for Production andQuality Control of Pneumococcal Conjugate Vaccines. Geneva, 4–5 June2003.

3. Training manual for enzyme linked immunosorbent assay for the quantitationof Streptococcus pneumoniae serotype specific IgG (Pn PS ELISA). Availableat: http://www.vaccine.uab.edu/

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4. Romero-Steiner S et al. Multilaboratory evaluation of a viability assay for themeasurement of opsonophagocytic antibodies specific to the capsularpolysaccharide of Streptococcus pneumoniae (Pnc). Clinical and DiagnosticLaboratory Immunology 2003, 10:1019–1024.

5. Quataert et al. Clinical and Diagnostic Laboratory Immunology. 1995, 2:590–597.

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 3Recommendations for the production and control ofinfluenza vaccine (inactivated)

Recommendations published by WHO are intended to be scientific andadvisory in nature. The parts of each section printed in type of normalsize have been written in a form, such that, should a national regulatoryauthority so desire, they may be adopted as they stand as definitivenational requirements or used as the basis of such requirements. Thoseparts of each section printed in small type are comments and recommen-dations for guidance for those manufacturers and national regulatoryauthorities which may benefit from additional information.

It is recommended that modifications be made only on condition that themodifications ensure that the vaccine is at least as safe and efficaciousas that prepared in accordance with the recommendations set out below.In order to facilitate the international distribution of vaccine made inaccordance with these recommendations, a summary protocol for therecording of results of the tests is given in Appendix 1.

The terms “national regulatory authority” and “national control laboratory”as used in these recommendations, always refer to the country in whichthe vaccine is manufactured.

Introduction 101

General considerations 102

Part A. Manufacturing recommendations 104A.1 Definitions 104A.2 General manufacturing requirements 107A.3 Production control 108A.4 Filling and containers 120A.5 Control tests on final lot 121A.6 Records 122A.7 Retained samples 122A.8 Labelling 122A.9 Distribution and transport 122A.10 Stability testing, storage and expiry date 123

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Part B. Requirements for national control authorities 123B.1 General 123B.2 Release and certification 124B.3 Clinical evaluation of influenza vaccines 124

Authors 124

Acknowledgements 125

References 125

Appendix 1Summary protocol for influenza vaccine (inactivated) (master/workingseed lot Type A or Type B) 127

Appendix 2Reference laboratories 133

Appendix 3Model certificate for the release of influenza vaccines (inactivated) 134

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Introduction

Influenza is a significant cause of morbidity and mortality and has amajor social and economic impact throughout the world. Duringmajor epidemics many people require medical treatment or hos-pitalization. Excess mortality often accompanies influenza epidemics,the vast majority of those affected being elderly. Because the elderlyconstitute the most rapidly increasing sector of the populationin many countries, the epidemiology of influenza can be expected tochange accordingly, especially in the developed countries. At present,the only means of influenza prophylaxis generally available isvaccination.

In 1967, a group of experts formulated requirements for inactivatedinfluenza vaccine and these were published as an annex to the twen-tieth report of the Expert Committee on Biological Standardization(1). During the following 5 years, technical developments in the puri-fication of the virus suspensions from which vaccines were made, aswell as in the measurement of the virus content, were such that thepotency of whole virus vaccines could be expressed in internationalunits. Accordingly, an addendum to the requirements was annexed tothe twenty-fifth report of the Expert Committee on Biological Stan-dardization (2). In its twenty-ninth report (3), the Committee recog-nized that technical developments had completely altered the methodof measurement of the haemagglutinin content of the vaccinesand that the International Reference Preparation of Influenza VirusHaemagglutinin (Type A) established in 1967 was no longer appro-priate for controlling the haemagglutinin content of inactivatedinfluenza vaccines because it no longer represented the haemag-glutinin of the prevalent strains. Accordingly, the InternationalReference Preparation was withdrawn, and the Committee recom-mended that the requirements for inactivated influenza vaccineshould be revised. Revised requirements were approved by the WHOExpert Committee on Biological Standardization in 1978 (4) andmodified in 1990 (5).

Since 1990, there have been significant new developments in methodsof influenza vaccine production resulting from: increased develop-ment of mammalian cell lines for vaccine production (6); increasedexperience in use of adjuvants; and rapid development of reversegenetics technologies for generation of vaccine viruses. There has alsobeen considerable effort directed to pandemic planning to ensure thatsafe, effective vaccines can be quickly produced in response to apandemic emergency. Consequently it has become necessary to revisethe requirements to reflect these new developments. In accordance

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with current WHO policy, the revised document is renamed as“Recommendations”.

General considerations

Inactivated influenza vaccines have been in widespread use for nearly60 years. The efficacy of immunization has varied according to cir-cumstances, but protection rates of 75–90% have been reported. Dif-ferences in protective efficacy may result from continuing antigenicvariation in the prevalent epidemic strains. Because of this variation,the composition of inactivated influenza virus vaccine, unlike that ofmost viral vaccines, must be kept constantly under review. Accord-ingly, WHO publishes recommendations concerning the strains to beincluded in the vaccine twice annually.

Influenza vaccines usually contain one or more influenza A viruses.However, because influenza A viruses undergo frequent and pro-gressive antigenic drift in their haemagglutinin and neuraminidaseantigens, vaccines containing formerly prevalent viruses are expectedto be less protective against virus variants showing antigenic drift thanagainst the homologous virus. When a new subtype of influenza Avirus bearing new haemagglutinin (and neuraminidase) antigen(s)appears, it is likely that vaccine containing the antigen(s) of theinfluenza A subtype(s) formerly prevalent will be ineffective, so thata vaccine containing the new pandemic virus will be required.

Changes in the structure of the haemagglutinin and neuraminidasemolecules, which result in changes in antigenicity as new epidemicstrains appear, involve surface residues in the region of the moleculefurthest from the viral envelope. Prediction of future variations is notpossible because the mechanism of selection of antigenic variants,antigenic drift, is not known and several evolutionary pathways ap-pear possible. Antigenic shift (i.e. the appearance of epidemic strainswith a new haemagglutinin subtype) is also unpredictable.

Antigenic drift in influenza B virus strains is less frequent than that inthe A strains and antigenic shift is unknown. Although distinct lin-eages of influenza B may occasionally co-circulate, it is usual forinfluenza vaccines to contain only one influenza B strain.

In addition to antigenic drift and shift, there is another type of varia-tion among influenza viruses, namely the preferential growth of cer-tain virus subpopulations in different host cells in which the virus iscultivated. Influenza viruses grown in embryonated eggs often exhibitantigenic and biological differences from those isolated and

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maintained in mammalian cells. Sequence analysis of the haemag-glutinin gene of such variants has shown that, typically, virus grown inmammalian cells differs from virus from the same source cultivated ineggs only by the substitution of a single amino acid in the haemag-glutinin molecule.

The WHO Expert Committee on Biological Standardization recom-mended in its twenty-ninth report (3) that the potency of influenzavaccines should be expressed inmg of haemagglutinin per ml (ordose), as determined by suitable immunodiffusion methods. In orderto standardize these methods, reference antigen (calibrated in mg ofthe haemagglutinin per ml) and specific anti-haemagglutinin serum,suitable for use in the assay of the haemagglutinin content of eachcomponent of inactivated vaccines, are prepared and distributed byreference laboratories (Appendix 2). A new reference antigen andantiserum is prepared each time it is necessary to introduce a newvirus strain into the vaccines, and these are standardized by interna-tional collaborative study.

Over the past 20 years there have been many clinical trials of wholevirus, split and subunit influenza vaccines. This has led to the gener-ally accepted view that one dose of vaccine containing 15mg ofhaemagglutinin per strain per dose, will stimulate haemagglutination-inhibition antibody levels consistent with immunity in most primedindividuals (7).

There has been much progress in developing influenza vaccines withadjuvant and some such vaccines are now licensed. The main issuesfor vaccine quality are: demonstration of compatibility of the adju-vant with the antigenic components of the vaccine; proof of consistentassociation with vaccine antigens (if appropriate) at time of produc-tion and throughout shelf-life; effect of adjuvant on vaccine potencyassays; and biochemical purity of adjuvant.

There is a long history of safety for egg-grown vaccines. However it isknown that influenza viruses cultivated in eggs can be contaminatedwith other viral agents and there has been a recent example of con-tamination of a candidate pandemic vaccine virus with avian adenovi-rus. These recommendations have therefore been revised in view ofthe findings with egg-grown viruses, the increasing use of mammaliancells for virus isolation and vaccine production, and the improvedmethods for detecting extraneous agents.

Influenza pandemic alerts occurred in 1997 (H5N1 virus), 1999 (H9N2virus) and 2003 (H5N1 and H7N7 viruses), when avian influenzaviruses caused serious illness and, on occasion, death in humans.

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Experience gained from these events has illustrated that differentstrategies for the production and clinical use of vaccine may beneeded in response to a pandemic.

• It may be necessary to generate a vaccine virus from a highlypathogenic virus by use of reverse genetics.

• Initially, reference reagents for testing vaccine potency may not beavailable;

• A vaccine with an adjuvant may be desirable.• Monovalent vaccine may be preferred to conventional trivalent

vaccine.• Whole virus vaccine may be preferred to a split or subunit

preparation.• A different dosing strategy may be needed (i.e. two doses).

It is important to develop WHO recommendations for productionand quality control of the vaccine that reflect the special needs of apandemic.

Although the technology is still under development, the use of re-verse genetics for vaccine virus development is likely to affectinterpandemic and pandemic vaccines alike. This technology involvestransfecting mammalian cells with plasmids coding for influenzavirus genes in order to produce a virus reassortant. Production ofreassortants by reverse genetics is similar in concept to traditionalmethods, but there are some quality issues which should be taken intoaccount.

• The influenza virus haemagglutinin and neuraminidase genes maybe derived from a variety of sources (an egg isolate, an isolate incells approved or not approved for human vaccine production,virus present in clinical specimens).

• The reassortant virus will have been generated in mammalian cells.• In some countries, reassortants produced by reverse genetics may

be classified as “genetically modified organisms” and vaccine pro-duction should comply with national Contained Use regulations(although the final inactivated product will not be a geneticallymodified organism).

Part A. Manufacturing recommendations

A.1 DefinitionsA.1.1 Proper name

The proper name shall be “influenza vaccine (whole virion,inactivated)” or “influenza vaccine (split virion, inactivated)”,

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“influenza vaccine (surface antigens, inactivated)” or “influenza vac-cine (inactivated, adjuvanted)” translated into the language of thecountry of use.

The use of the proper name should be limited to vaccines that satisfy therecommendations specified below.

A.1.2 Descriptive definition

Influenza vaccine is a sterile, aqueous suspension of a strain or strainsof influenza virus, type A or B, or a mixture of these two types, whichhave been grown individually in embryonated hen’s eggs or in mam-malian cells. Four types of influenza vaccine are available:

(i) a suspension of whole virus particles inactivated by a suitablemethod;

(ii) a suspension treated so that the virus particles have beenpartially or completely disrupted by physicochemical means(split vaccine);

(iii) a suspension treated so that the preparation consists predomi-nantly of haemagglutinin and neuraminidase antigens (subunitvaccine);

(iv) a suspension of inactivated influenza virus particles, split or sub-unit components formulated with an adjuvant.

The preparation should satisfy all the requirements formulated below.

A.1.3 Choice of vaccine strain

The World Health Organization reviews the world epidemiologicalsituation twice annually and if necessary recommends new vaccinestrain(s) in accordance with the available evidence.

Such strains, or those antigenically related to them, should be usedin accordance with the regulations in force in the countryconcerned.

It is now common practice to use reassortant strains that give high yields ofthe appropriate surface antigens. Reassortant strains for vaccine produc-tion have the surface glycoproteins (haemagglutinin and neuraminidase) ofthe recommended reference virus and the internal proteins of a high-growthdonor virus.

These recommendations shall also apply to the subsequent productionand quality control of reassortant vaccine viruses produced by reversegenetics.

The passage history of the parent and reassortant virus strains shouldbe approved by the national regulatory authority.

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A.1.4 Reference reagents

WHO reference antigens for strain characterization of influenza virusare preparations that are antigenically representative of viruses iso-lated throughout the world. They may be obtained from one of theWHO Collaborating Centres for Reference and Research on Influ-enza (see Appendix 2).

Candidate influenza vaccine viruses are preparations antigenicallyrepresentative of a virus strain likely to be included in a currentvaccine. They may be wild-type viruses or reassortant viruses withsurface antigens appropriate for the current recommendations forvaccine strains. They are distributed on demand when a new virusappears and the likelihood of its spreading throughout the worldmakes its inclusion in a vaccine desirable. These preparations may beobtained from one of the custodian laboratories listed in Appendix 2.

Antigen reagents for standardization of vaccine potency contain acalibrated quantity of haemagglutinin antigen of influenza virus mea-sured in mg/ml. The calibrations are performed by international col-laborative study using single radial immunodiffusion tests (8) withpurified virus of known haemagglutinin antigen concentration. Thereference antigen and antiserum reagents are used to calibrate thehaemagglutinin content of inactivated influenza vaccines by an invitro immunodiffusion test. These reference haemagglutinin antigens,together with the specific antihaemagglutinin sera, may be obtainedfor the purpose of such tests from one of the custodian laboratorieslisted in Appendix 2.

A.1.5 Terminology

Master seed lot: A quantity of virus, antigenically representative of aWHO-recommended strain, that has been processed at one time toassure a uniform composition and is fully characterized. It is used forthe preparation of working seed lots. The master seed lot and itspassage level are approved by the national authority.

Working seed lot: A quantity of fully characterized virus of uniformcomposition that is derived from a master seed lot by a number ofpassages that does not exceed the maximum approved by the nationalregulatory authority. The working seed lot is used for the productionof vaccines.

Cell seed: A quantity of well-characterized cells of human or animalorigin stored frozen in liquid nitrogen in aliquots of uniform compo-sition derived from a single tissue or cell, one or more of which wouldbe used for the production of a master cell bank.

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Master cell bank: A quantity of fully characterized cells of human oranimal origin derived from the cell seed stored frozen in liquid nitro-gen in aliquots of uniform composition, one or more of which may beused for the production of a manufacturer’s working cell bank. Thetesting performed on a replacement master cell bank (derived fromthe same clone or from an existing master or working cell bank) is thesame as for the initial master cell bank, unless a justified exception ismade.

Working cell bank (WCB): A quantity of cells of uniform compositionderived from one or more ampoules of the master cell bank, whichmay be used for the production cell culture. In normal practice, a cellbank is expanded by serial subculture up to a passage number (orpopulation doubling, as appropriate) selected by the manufacturer, atwhich point the cells are combined to give a single pool and preservedcryogenically to form the manufacturer’s WCB. One or more of theampoules from such a pool may be used for the production cellculture.

Production cell culture: A cell culture derived from one or moreampoules of the manufacturer’s WCB and used for production of thelive influenza virus.

Single harvest: A quantity of virus suspension derived from either agroup of embryonated eggs or a culture of mammalian cells that wereinoculated with the same virus working seed lot, incubated togetherand harvested together in one session.

Monovalent virus pool: A pool of a number of single harvests of asingle virus strain processed at the same time.

Final bulk: The finished vaccine prepared from one or more monova-lent pools present in the container from which the final containers arefilled. It may contain one or more virus strains.

Final lot: A collection of sealed final containers that are homogeneouswith respect to the risk of contamination during filling. A final lotmust therefore have been filled in one working session from a singlefinal bulk.

A.2 General manufacturing requirements

The general requirements for manufacturing establishments con-tained in good manufacturing practices for pharmaceuticals (9) andbiological products (10) should apply to establishments manufacturing inactivated influenza vaccine, with the addition of thefollowing:

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Details of standard operating procedures for the preparationand testing of influenza vaccines adopted by a manufacturer togetherwith evidence of appropriate validation of the production process,should be submitted for approval to the national regulatory authority.Proposals for modification of the manufacturing/control methodsshould also be submitted for approval by the national regulatoryauthority.

Personnel employed in the production and control facilities should beadequately trained and protected against accidental infection withinfluenza virus (11).

High levels of biological containment are likely to be required for theproduction of vaccines for use in pandemic or potential pandemicsituations. WHO will provide advice where appropriate (12) andnational or regional safety guidelines must be followed.

Standard operating procedures need to be developed for dealing withemergencies involving accidental spillage, leakage or other dissemi-nation of influenza virus.

The areas where processing of inactivated influenza vaccine takesplace shall be separate from those where work with live influenzavirus is performed.

A.3 Production controlA.3.1 Control of source materialsA.3.1.1 Eggs used for seed virus growth

If the vaccine is to be produced in embryonated eggs, the eggs to beused should be from closed, specific-pathogen-free, healthy flocks.

The flock should be monitored at regular intervals for specific agents. Theagents monitored may include Mycobacterium avium, fowlpox virus, avianleukosis virus (ALV) and other avian retroviruses, Newcastle disease virusand other avian parainfluenza viruses, avian encephalomyelitis virus,infectious laryngotracheitis virus, avian reticuloendotheliosis virus, Marek’sdisease virus, infectious bursal disease virus, Haemophilus paragallinarum,Salmonella gallinarum, Salmonella pullorum, Mycoplasma gallisepticumand Mycoplasma synoviae.

In some countries, all birds are bled when a colony is established, andthereafter 5% of the birds are bled each month. The resulting serumsamples are screened for antibodies to the relevant pathogens. Any birdthat dies should be investigated to determine the cause of death.

A.3.1.2 Eggs used for vaccine productionIf the vaccine is to be produced in embryonated eggs, the eggs shouldbe from healthy flocks, which are monitored by methods approved bylocal animal health authorities.

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As large numbers of eggs are needed for vaccine production, it is notfeasible to use eggs from specific-pathogen-free flocks.

Monitoring of flocks for avian influenza viruses is performed in somecountries.

In both situations (production of vaccine seed and production ofvaccine), the flock must not have been vaccinated with live Newcastledisease virus vaccine. It is also recommended that eggs be obtainedfrom young birds.

In countries where use of live Newcastle disease vaccine is mandatory,vaccination should take place during the first few weeks of the chickens’ lifeand well before the use of flocks for supply of eggs.

A.3.1.3 Master cell bank and manufacturer’s working cell bankIf a cell line is used for the manufacture of influenza vaccines, itshould be based on the cell bank system. The national regulatoryauthority should approve the master cell bank and should establishthe maximum number of passages (or population doublings) by whichthe manufacturer’s WCB is derived from the master cell bank and themaximum number of passages of the production cultures.

WHO has established a reference cell bank of Vero cells1 characterized inaccordance with the requirements produced in 1996 (13) as modified byAnnex 4 of this report (14). This should not be considered as a master cellbank for direct use in vaccine production, but may be used to develop amaster cell bank by thorough requalification.

A.3.1.3.1 Identity test

The master cell bank should be characterized according to the WHORequirements for the use of animal cells as in vitro substrates for theproduction of biologicals (13, 14) as they relate to continuous celllines, or to human diploid cells, as appropriate.

The manufacturer’s WCB should be identified by means, inter alia, ofbiochemical (e.g. isoenzyme analysis), immunological and cytogeneticmarker tests, and DNA fingerprinting, approved by the national regu-latory authority.

A.3.1.4 Cell culture mediumSerum used for the propagation of cells should be tested to demon-strate freedom from bacteria, fungi and mycoplasmas, according tothe requirements given in sections A.5.2 and A.5.3 of the revised

1 Available to manufacturers on application to Quality Assurance and Safety ofBiologicals: Vaccines and Biologicals, World Health Organization, 1211 Geneva 27,Switzerland.

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Requirements for Biological Substances No. 6 (15) and from infec-tious viruses. Suitable tests for detecting viruses in bovine serum aregiven in Appendix 1 of the Recommendations for PoliomyelitisVaccine (Oral) (16).

Where approved by the national regulatory authority, alternative tests forbovine viruses may be used.

As an additional monitor of quality, sera may be examined for freedom fromphage and endotoxin.

Irradiation may be used to inactivate potential contaminant viruses.

The sources(s) of serum of bovine origin should be approved by thenational regulatory authority. The serum should comply with currentguidelines in relation to animal transmissible spongiform encephalo-pathies (17).

Human serum should not be used. If human albumin is used, it shouldmeet the revised Requirements for Biological Substances No. 27(Requirements for the Collection, Processing and Quality Control ofBlood, Blood Components and Plasma Derivatives) (18), as well ascurrent guidelines in relation to human transmissible encephalopa-thies (17).

Manufacturers are encouraged to explore the possibilities of using serum-free media for production of inactivated influenza vaccine.

Penicillin and other b-lactams should not be used at any stage of themanufacture.

Other antibiotics may be used at any stage in the manufacture, providedthat the quantity present in the final product is acceptable to the nationalregulatory authority. Nontoxic pH indicators may be added, e.g. phenol redin a concentration of 0.002%. Only substances that have been approved bythe national regulatory authority may be added.

Trypsin used for preparing cell cultures should be tested and foundfree of cultivable bacteria, fungi, mycoplasmas and infectious viruses,especially parvoviruses appropriate to the species of animal used. Themethods used to ensure this should be approved by the nationalregulatory authority.

The source(s) of trypsin of bovine origin, if used, should be approvedby the national regulatory authority. Bovine trypsin, if used, shouldcomply with current guidelines in relation to animal transmissiblespongiform encephalopathies (17).

A.3.1.5 Virus strainsStrains of influenza virus used in the production of inactivated influ-enza vaccine should be identified by historical records, which should

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include information on the origin of the strains and their subsequentmanipulation.

Only strains that have been isolated in embryonated hen’s eggs,in cells derived from eggs, or in mammalian cells approved forhuman vaccine production under validated laboratory conditionsshould be used. The national regulatory laboratory should approvethe virus strain. It is now common practice to use reassortant strainsgiving high yields of the appropriate surface antigens. However, ithas been noted that antigenic changes may occur during the deve-lopment of high-yielding reassortants, and the absence of suchchanges should be shown by haemagglutination-inhibition testsusing antibodies to the haemagglutinin of the reassortant and of wildtype-viruses.

Where reassortant strains are used, the parent high-yield strain andthe method of preparing the reassortant should be approved by thenational regulatory authority.

Where reverse genetics is used to generate the reassortant vaccine virus,the influenza haemagglutinin and neuraminidase genes may be derivedfrom a variety of sources (egg isolate, mammalian cell isolate or virus inclinical specimen). The haemagglutinin and neuraminidase genes areexpected to be free of extraneous agents associated with the wild-typevirus by virtue of the recombinant DNA technology employed. The cellsubstrate used for transfection to generate the reassortant virus should beapproved for human vaccine production. The derivation of the reassortantvirus should be approved by the national regulatory authority.

Strains of virus suitable for manufacture of a vaccine for use in a pandemicor potential pandemic should be supplied by procedures agreed by theWHO Collaborating Centres for Reference and Research on Influenza andthe custodian laboratories for supply of candidate strains (Appendix 1) andapproved by the national regulatory authority.

If any materials of animal (non-avian) origin are used in production,they should comply with the guidance given in the report of a WHOConsultation on medicinal and other products in relation to humanand animal transmissible spongiform encephalitis (17) and should beapproved by the national regulatory authority.

Reference strains for antigenic analysis may be obtained from the WHOCollaborating Centres for Reference and Research on Influenza, or othercustodian laboratories (Appendix 2).

A.3.1.5.1 Seed lot system

The production of vaccine should be based on a seed lot system. Eachseed lot should be identified as influenza virus of the appropriatestrain by methods acceptable to the national regulatory authority(section A.1.3). The maximum number of passages between a masterseed lot and a working seed lot should be approved by the national

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regulatory authority. The vaccine should be not more than one pas-sage from the working seed lot.

Each manufacturer should identify the haemagglutinin andneuraminidase antigens of the vaccine virus strains by suitable testscapable of detecting biologically significant variation, as well as cross-contamination during manipulation.

A.3.1.5.2 Tests on seed lots

Either the master or working seed virus should be shown to be freefrom relevant extraneous agents by tests or procedures approved bythe national regulatory authority in accordance with the requirementsof Part A, sections 5.2 and 5.3, of the revised Requirements forBiological Substances No. 6 (General Requirements for the Sterilityof Biological Substances) (15).

Strategies to ensure freedom from extraneous agents in the final vaccinemay involve a combination of testing the seed virus and validation of theproduction process, depending on the substrate used for production. Foregg-derived vaccines, the emphasis should be on a validation process,whereas for cell-derived vaccines, the emphasis may be on a testingstrategy. The national regulatory authority should approve the strategychosen.

Validation strategyThe production process should be validated to demonstrate removal and/orinactivation of likely potential contaminating agents. Validation may beperformed using appropriate model agents.

If removal or inactivation cannot be demonstrated for a potentialcontaminant, a testing strategy should be implemented.

Testing strategy

Cell-derived vaccineThe susceptibility of mammalian cells to various human pathogens shouldbe taken into account and this information should be used in considering alist of potential human pathogens to be included in testing for extraneousagents in seed virus. Pathogens to be considered could includeadenovirus, parainfluenza virus, respiratory syncytial virus, coronavirus,rhinovirus, enterovirus, human herpesvirus 4 (Epstein–Barr virus), herpessimplex virus, cytomegalovirus and mycoplasmas.

It is recognized that when a vaccine strain changes, there may be timeconstraints that make testing seed viruses for extraneous agentsproblematic, and the full results of such testing may not always be availablebefore further processing. The use of rapid assays (e.g. multiplexpolymerase chain reaction (PCR)) which could be applied within these timeconstraints is encouraged.

If an extraneous agent is detected in a seed virus and the mammalian cellsused for production are shown to be susceptible to this agent, the seedvirus should not be used for vaccine production.

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If an extraneous agent is detected in a seed virus and the mammaliancells are not susceptible to the agent, steps should be in place to ensurethat the contaminating agent is removed and/or inactivated by theproduction process. If removal or inactivation of the agent cannot bedemonstrated, appropriate and specific downstream testing at the levelof each inactivated monovalent bulk should be implemented to demon-strate that any contaminant identified in the seed virus is absent from thevaccine.

Egg-derived vaccine

A strategy for testing for specific potential contaminating agents may beneeded if removal or inactivation of the agent by the production processcannot be demonstrated. The use of rapid tests (e.g. PCR) is encouraged.If the agent is detected, the seed virus should not be used for vaccineproduction.

The seed lot should be stored at a temperature lower than -60°C,unless it is in the lyophilized form, in which case it should be stored ata temperature lower than -20°C.

A.3.2 Production precautions

The manufacture of inactivated influenza vaccines should follow therelevant guidance on good manufacturing practice (10, Annex 1) andquality assurance (10, Annex 2) for biological products with the addi-tion of the following:

• for egg-derived vaccines, only allantoic and amniotic fluids may beharvested.

• b-Lactam antibiotics should not be used at any stage in the manu-facture of the vaccine.

Minimal concentrations of other suitable antibiotics may be used.

If vaccines are produced by the splitting of the virus by chemicalmeans, the splitting conditions and the concentration of the chemicalsused should be approved by the national regulatory authority. If anadjuvant is used, the concentration of adjuvant should be approved bythe national regulatory authority.

A.3.3 Production of monovalent virus poolsA.3.3.1 Single harvests

For egg-derived vaccine, each strain of virus should be grown in theallantoic cavity of embryonated hen’s eggs derived from healthyflocks. After incubation at a controlled temperature, both the allan-toic and amniotic fluids may be harvested. For mammalian-cell de-rived vaccine, each strain of virus should be grown in cells approvedfor human vaccine production.

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For both cell-derived and egg-derived vaccines, a number of singleharvests of the same strain of virus may be combined to give amonovalent virus pool. Cell-derived monovalent virus pools shouldnot be mixed with egg-derived monovalent virus pools.

A.3.3.2 Inactivation of monovalent virus poolsTime of inactivation. To limit the possibility of contamination,monovalent virus pools should be inactivated as soon as possible aftertheir preparation. However, if delay is unavoidable, the temperatureand duration of the storage should be validated with respect tobioburden and quality of the haemagglutinin and neuraminidaseantigens.

Validation with respect to bioburden may be omitted for cell-culture-derivedmonovalent virus pools, with the agreement of the national regulatoryauthority.

Before monovalent virus pools are inactivated, samples shall betaken and tested for bacterial and fungal contamination. Limitsfor bioburden should be approved by the national regulatoryauthority.

Inactivation procedure. The virus in the monovalent virus poolsshould be inactivated by a method that has been demonstrated to beconsistently effective in the hands of the manufacturer and has beenapproved by the national regulatory authority. For egg-derived vac-cine, the inactivation process should also have been shown, to thesatisfaction of the national regulatory authority, to be capable ofinactivating avian leukosis viruses and mycoplasmas. If the virus poolis stored after inactivation, the temperature and duration of the stor-age should be validated. The inactivation procedure should have beenshown to be capable of inactivating influenza viruses without destroy-ing their antigenicity.

The usual storage temperature is 5 °C ± 3 °C.

Consideration should be given to investigating whether influenza virusinactivation also inactivates human or avian pathogens capable of be-coming extraneous agents. These investigations may be performed usingappropriate model agents e.g.

— egg-derived vaccines: avian leukosis virus, mycoplasma, avianadenovirus;

— cell-derived vaccines: poliovirus, human immunodeficiency virus,human adenovirus, parainfluenza virus, minute virus of mice.

If formalin (40% formaldehyde) or b-propiolactone (2-oxetanone) isused, the concentration by volume should not exceed 0.1% at anytime during inactivation. Other suitable inactivating agents can also beused.

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Consideration should be given to strategies to limit the entry into themanufacturing process of potential adventitious agents that may not beinactivated by the influenza inactivation conditions.

A.3.3.3 Testing of control cellsA cell sample equivalent to at least 500ml of the cell suspension, atthe concentration employed for vaccine production cultures, shouldbe used to prepare control cell cultures. In countries with the technol-ogy for large-scale production, the national regulatory authorityshould determine the size of the sample of cells to be examined, thetime at which the control cells should be taken from the productionculture, and how the control cells are maintained.

These control cell cultures should be incubated for at least 2 weeksand should be examined during this period for evidence of cytopathicchanges. For the test to be valid, not more than 20% of the controlcell cultures may have been discarded for nonspecific, accidentalreasons.

If this examination or any of the tests required in this section showevidence of the presence in a control culture of any adventitiousagent, the influenza virus grown in the corresponding inoculated cul-tures should not be used for vaccine production.

Samples not tested immediately should be stored at -60°C or below.

A.3.3.3.1 Tests for haemadsorbing viruses

At the end of the observation period or at the time the virus isharvested from the production cultures, whichever is the later, at least25% of the control cells should be tested for the presence ofhaemadsorbing viruses using guinea-pig red blood cells. If the cellshave been stored, the duration of storage should not have exceeded 7days, and the storage temperature should have been in the range of2–8 °C. In tests for haemadsorbing viruses, calcium and magnesiumions should be absent from the medium.

This test is usually done using guinea-pig red cells. However, in somecountries the national regulatory authority requires that additional tests forhaemadsorbing viruses should be made in other types of red cell, includingthose from humans (blood group O), monkeys and chickens (or other avianspecies).

The results of all tests should be read after incubation for 30min at 0–4 °Cand again after a further incubation for 30 minutes at 20–25°C. A furtherreading for the test with monkey red blood cells should also be taken afteranother incubation for 30min at 34–37 °C.

In some countries the sensitivity of each new batch of red blood cells isdemonstrated by titration against a haemagglutinin antigen before use inthe haemadsorbtion test.

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A.3.3.3.2 Tests on supernatant fluids

A sample of at least 10 ml of the pooled supernatant fluid from thecontrol cultures collected at the end of the observation period shouldbe tested in the same cell substrate, but not the same batch, as thatused for production. Additional samples of at least 10ml should betested in both human and monkey cells. The samples should be inocu-lated into bottles of these cell cultures, in such a way that the dilutionof the supernatant fluid in the nutrient medium does not exceed 1 in4. The area of the cell sheet should be at least 3cm2/ml of supernatantfluid. At least one bottle of each of the cell cultures should remainuninoculated and serve as a control.

The cultures should be incubated at a temperature of 35–37°C andshould be observed for a period of at least 2 weeks.

The use of rapid assays (e.g. multiplex PCR), which could be conductedwithin the time constraints of the procedure are encouraged.

A.3.3.3.3 Identity test

For vaccines produced in continuous cell culture the controlcells should be identified by means, inter alia, of biochemical (e.g.isoenzyme analysis), immunological and cytogenetic marker testsapproved by the national regulatory authority.

A.3.3.4 Concentration and purificationThe monovalent material should be concentrated and purified byhigh-speed centrifugation or other suitable methods approved by thenational regulatory authority, either before or after the inactivationprocedure.

The aim is to separate the virus or viral components from other constituentsin either the allantoic and amniotic fluids or the mammalian cell culture fluidsas efficiently as possible. It is advisable to concentrate and purify the virusunder optimum conditions to preserve its antigenic properties.

Consideration should be given to investigating whether the concentrationand purification steps remove potential extraneous agents.

A.3.4 Control of monovalent virus poolsA.3.4.1 Effective inactivation

The inactivated and purified monovalent virus pool should be shownnot to contain viable influenza virus when tested by a method ap-proved by the national regulatory authority. Tests for viable virusshould be conducted in eggs for egg-derived vaccine and in the mam-malian cells used for vaccine production for cell-derived vaccine.

A suitable method for egg-derived vaccine consists of inoculating 0.2ml ofundiluted monovalent pool and 1 :10 and 1 :100 dilutions of the monovalentpool into the allantoic cavities of groups of fertilized eggs (ten eggs in each

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group), and incubating the eggs at 33–37°C for 3 days. At least eight of theten embryos should survive at each dosage level.

A volume of 0.5ml of allantoic fluid is harvested from each surviving egg. Thefluid harvested from each group is pooled and 0.2ml of each of the threepools is inoculated, undiluted, into a further group of ten fertile eggs.Haemagglutinin activity should not be detected in these new groups of eggs.

In some countries, alternative methods are used.

In some countries, the requirement that 80% of the embryos should surviveduring incubation may be impossible to satisfy. The national regulatoryauthority should then specify the requirement to be satisfied.

For mammalian-cell-derived vaccine, methods could be modelled on thoseused for egg-derived vaccines with the exception that a mammalian cellsubstrate with validated sensitivity should be used. The national regulatoryauthority should approve the method used and specify the requirement tobe satisfied.

As testing of residual virus infectivity after inactivation may be problematicdue to aggregation, validation of test sensitivity should be performed.

A.3.4.2 Haemagglutinin contentThe content of haemagglutinin in the monovalent virus pool shouldbe determined by a suitable and approved technique, such as singleradial immunodiffusion. In the test, an influenza referencehaemagglutinin antigen reagent or a national preparation calibratedagainst it should be used for purposes of comparison (see sectionA.1.4).

For adjuvanted vaccines it should be established whether the adjuvant iscompatible with the antigenic components of the vaccine and whether thepresence of adjuvant interferes with the test for haemagglutinin content. Ifthere is likely to be interference, this test may be performed before theaddition of adjuvant. The test should be fully validated.

There is evidence to suggest that when cell-derived vaccines are producedfrom viruses isolated in eggs, the conventional reference antigen reagents(egg-derived) are suitable for measurement of haemagglutinin content.However the use of conventional antigen reagents may not be suitable tomeasure the haemagglutinin content of mammalian cell-derived vaccineswhen viruses isolated in cells are used for production. As further informationbecomes available, advice will be provided by WHO.

During the early stages of production of vaccines for pandemics, theremay be no reference reagents to measure vaccine haemagglutinincontent by conventional methods. It may be necessary to use alternativeestimates of antigen content as advised by WHO and national regulatoryauthorities.

A.3.4.3 Presence of neuraminidaseVaccine should be prepared under conditions that allow retention ofdetectable levels of viral neuraminidase for each strain.

In some countries, a test is included for the presence of neuraminidaseenzymatic or antigenic activity. The ratio of haemagglutinin to neurami-

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nidase should be consistent for the particular virus strain and method ofvaccine production used, but the neuraminidases of different strains varymarkedly in their stability during processing.

A.3.4.4 Virus disruption (split vaccines)Monovalent pools in which the virus has been split by chemical meansshould be shown by procedures approved by the national regulatoryauthority to consist predominantly of disrupted virus particles.

This test need be performed on only three samples of monovalent pool foreach vaccine strain provided that the test result is satisfactory.

A.3.4.5 Surface antigens (subunit vaccines)The purity of monovalent pools intended for the preparation ofsubunit vaccine shall be determined by polyacrylamide gel electro-phoresis or by other suitable techniques approved by the nationalregulatory authority. Mainly haemagglutinin and neuraminidaseantigens for each strain should be present.

This test need be performed on only three samples of monovalent pool foreach vaccine provided that the test result is satisfactory.

A.3.4.6 IdentityAntigenic specificity may be confirmed by an immunodiffusion orhaemagglutination-inhibition technique using appropriate specificimmune sera. The tests for haemagglutinin content (A.3.4.2) andpresence of neuraminidase (A.3.4.3) also serve as identity tests.Reference viruses for identity tests may be obtained from referencelaboratories (Appendix 2).

Alternatively antigenic identity may be confirmed by:

— injection of vaccine into mice, chickens or other suitable animals anddemonstration of the production of antibodies to the haemagglutinin ofthe influenza virus used to produce the vaccine. In addition,demonstration of production of antibody to neuraminidase may also beperformed; or

— suitable genetic tests.

With split and subunit vaccines, the identity test may be performedbefore virus disruption.

A.3.4.7 Extraneous agents

Cell-derived vaccinesIf a contaminating agent is found in the working seed, mammaliancells are not susceptible to infection by the agent (A.3.1.5.2) andremoval and/or inactivation of the agent by the production processcannot be demonstrated, monovalent bulks should be tested to ensurefreedom from the agent. The data should be approved by the nationalregulatory authority.

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Egg-derived vaccinesIf removal and/or inactivation of a potential contaminating agent bythe production process cannot be demonstrated, monovalent bulksshould be tested to ensure freedom from the agent. The data shouldbe approved by the national regulatory authority.

A.3.4.9 Purity of cell-derived vaccinesTo monitor consistency in purity, monovalent virus pools derivedfrom mammalian cell cultures should be tested for the ratio ofhaemagglutinin content: total protein. This ratio should be within thelimits approved by the national regulatory authority.

For viruses grown in continuous cell culture, the purified monovalentpool should be tested for residual cellular DNA. The purificationprocess should be shown to consistently reduce the level of cellularDNA to less than 10ng per human dose. This test may be omitted,with the agreement of the national regulatory authority, if the manu-facturing process is validated as achieving this specification.

A.3.4.9 Tests for chemicals used in productionThe concentration of each detergent, organic solvent and inactivatingagent remaining in the final vaccine should be determined using meth-ods approved by the national regulatory authority. These concentra-tions should not exceed the upper limits specified by the nationalregulatory authority. For preservatives, both the method of testingand the concentration should be approved by the national regulatoryauthority.

Alternatively, tests for chemicals may be performed on the final bulk.

A.3.5 Control of final bulk

Final bulks are prepared by mixing and diluting monovalent pools ofthe relevant strains. In the preparation of the final bulk, only preser-vatives or other substances, including diluents, approved by the na-tional regulatory authority should be added. Such substances shouldhave been shown by appropriate tests not to impair the safety oreffectiveness of the product in the concentrations used, and shouldnot be added before samples have been taken for any tests that wouldbe affected by their presence.

Vaccines for use during pandemics are likely to contain only one strain.

A.3.5.1 Test for content of haemagglutinin antigenThe haemagglutinin concentration in the final bulk should be deter-mined as described in section A.3.4.2.

This test may be omitted if such a test is performed on each final lot.

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A.3.5.2 Sterility testsEach bulk should be tested for sterility by a method approved by thenational regulatory authority.

Many countries have regulations governing sterility testing. Wherethese do not exist, the revised Requirements for Biological Sub-stances No. 6 (General Requirements for the Sterility of BiologicalSubstances) (15) should be satisfied. If a preservative has been addedto the vaccine, appropriate measures should be taken to prevent itfrom interfering with the sterility test.

A.3.5.3 Total proteinThe total protein content should be not more than six times the totalhaemagglutinin content of the vaccine, as determined in the test forhaemagglutinin content, but in any case not more than 100mg ofprotein per virus strain per human dose and not more than a total of300 mg of protein per human dose.

In some countries, protein stabilizers are added to vaccine. The totalprotein content should reflect such additions. For subunit vaccines, a lowerprotein content is achievable, i.e. not more than 40mg of protein per virusstrain per human dose and not more than a total of 120mg of protein perhuman dose.

A.3.5.4 Ovalbumin (egg-derived vaccines)The ovalbumin content should be not more than 5mg per human dose.The amount of ovalbumin should be determined by a suitable tech-nique using a suitable reference preparation of ovalbumin.

Values of less than 1mg of ovalbumin per human dose are attainable andlower limits may be set.

A.3.5.4 Adjuvant contentIf an adjuvant has been added to the vaccine, its content should bedetermined by a method approved by the national regulatory author-ity. The amount and nature of the adjuvant should be within the rangeshown to be clinically effective and should be approved by thenational regulatory authority.

The formulation of adjuvant and antigen should be stable and consistent.The purity of the adjuvant should be demonstrated to be within the rangefound for vaccine lots shown to be clinically effective.

A.4 Filling and containers

The requirements concerning filling and containers given in Goodmanufacturing practices for biological products (10, annex 1, section4) should apply. Single- and multiple-dose containers may be used. Ifthe latter are used, a suitable preservative, approved by the nationalregulatory authority, should be incorporated.

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A.5 Control tests on final lotA.5.1 Identity test

An identity test should be performed by a method approved by thenational regulatory authority on at least one container from each finallot.

The identity of the haemagglutinins in the vaccine should be deter-mined by an immunological technique, such as immunodiffusion orhaemagglutinin inhibition, using the appropriate specific immuneserum.

In some countries, a test to identify the specific neuraminidase antigens isalso included.

A.5.2 Sterility test

Final containers should be tested for sterility as described in sectionA.3.5.2.

A.5.3 Haemagglutinin content

The test for haemagglutinin antigen concentration is performed asdescribed in section A.3.4.2.

The vaccine should contain in each human dose at least 15mg ofhaemagglutinin of each strain used in the preparation.

In some countries, lower limits may be set, based on clinical experience.

Expression of haemagglutinin antigen content can also reflect uncertainty ofmeasurement by stipulating that the lower confidence interval (P = 0.95) ofthe assay should be not less than 12mg of haemagglutinin of each strain perdose.

It may be necessary to formulate vaccine for use in a pandemic to containa different haemagglutinin antigen concentration. Advice will be providedby WHO and the national regulatory authority.

A.5.4 General safety (innocuity) tests

Each filling lot should be tested for unexpected toxicity (sometimescalled abnormal toxicity) using a general safety (innocuity) test ap-proved by the national regulatory authority.

This test may be omitted for routine lot release once consistency ofproduction has been well established to the satisfaction of the nationalregulatory authority and when good manufacturing practices are in place.Each lot, if tested, should pass a test for abnormal toxicity.

A.5.5 Endotoxin

A test for endotoxin should be included, e.g. the Limulus amoebocytelysate test.

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The permissible level of endotoxin is determined by the national regulatoryauthority. It is likely that the permissible level of endotoxin for mammalian-cell-derived vaccine will be lower than that for egg-derived vaccine.

A.5.6 Inspection of final containers

Each container in each final lot shall be inspected visually, and thoseshowing abnormalities such as lack of integrity shall be discarded.

A.6 Records

The requirements given in section 8 of Good manufacturing practicesfor biological products (10, annex 1) should apply.

A.7 Retained samples

The requirements given in section 9 of Good manufacturing practicesfor biological products (10, annex 1) should apply.

A.8 Labelling

The requirements given in section 7 of Good manufacturing practicesfor biological products (10, annex 1) should apply, with the additionof the following information.

The label on the carton, the container or the leaflet accompanying thecontainer should state:

— that the vaccine has been prepared from virus propagated inembryonated hen’s eggs or in mammalian cells;

— the type of cell line i.e. monkey, dog, etc. (if appropriate);— the strain or strains of influenza virus present in the preparation;— the haemagglutinin content in mg per virus strain, expressed as mg of

haemagglutinin per dose;— the number of doses, if the product is issued in a multiple-dose

container;— the influenza season for which the vaccine is intended;— the method used for inactivating the virus;— the name and maximum quantity of any antibiotic present in the vaccine;— the name and concentration of any preservative added;— the name and concentration of any adjuvant added;— the temperature recommended during storage and transport;— the expiry date; and— any special dosing schedules (e.g. for a pandemic vaccine).

For a pandemic vaccine — special dosing schedules (e.g. two doses).

A.9 Distribution and transport

The requirements given in section 8 of Good manufacturing practicesfor biological products (10, annex 1) should apply.

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A.10 Stability testing, storage and expiry dateA.10.1 Stability testing

Adequate stability studies form an essential part of vaccine deve-lopment. The stability of the vaccine in its final form and at therecommended storage temperatures should be demonstrated to thesatisfaction of the national regulatory authority on final containersfrom at least three lots of final product.

Stability data may be presented to the national regulatory authority after useof vaccine. Accelerated stability studies may be used.

In some countries, vaccine haemagglutinin content should comply with finalproduct specifications (see A.5.3) at the expiry date.

The formulation of vaccine antigens and adjuvant (if used) must bestable throughout its shelf-life. Acceptable limits for stability shouldbe agreed with national authorities.

When any changes are made in the production process that may affectstability of the products, the vaccine produced by the new methodshould be shown to be stable.

A.10.2 Storage conditions

Inactivated influenza vaccine should be stored at a temperature of2–8°C.

If other storage conditions are used, they should be fully validated andapproved by the national regulatory authority.

A.10.3 Expiry date

The expiry date should be fixed with the approval of the nationalregulatory authority, and should take account of the experimentaldata on stability of the vaccine.

In general, the expiry date should not exceed 1 year from the date of issueby the manufacturer because the strains used in one year’s vaccine maynot be appropriate the next year.

Part B. Requirements for national controlauthorities

B.1 General

The general recommendations for control laboratories, contained inthe Guidelines for national authorities on quality assurance of bio-logical products should apply (10, Annex 2).

The national regulatory authority should give directions to manufac-turers concerning the influenza virus strains to be used, the

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haemagglutinin content, whether or not neuraminidase is present,and the recommended human dose.

B.2 Release and certification

A vaccine lot shall be released only if it fulfils national requirementsand/or Part A of these Recommendations. A statement signed by theauthorized official of the national regulatory authority should be pro-vided at the request of the manufacturing establishment and shouldcertify that the lot of vaccine in question satisfies all national require-ments as well as Part A of these Recommendations. The certificateshould state the number under which the lot was released by thenational controller, and the number appearing on the labels of con-tainers. Importers of influenza vaccine (inactivated) should be given acopy of the official national release document. The purpose of thecertificate is to facilitate the exchange of inactivated influenza vaccinebetween countries.

An example of a suitable certificate is given in Appendix 3.

B.3 Clinical evaluation of influenza vaccines

In the case of a new manufacturer, the national regulatory authorityshould assess the safety and immunogenicity of the vaccine by arrang-ing for studies in human volunteers of one or more of the lots ofvaccine that have satisfied the above-mentioned requirements. Suchstudies shall include the assessment of the immune responses andadverse reactions in various age groups.

In the case of a significant change in the manufacturing process, clinicalstudies may also be required by the national regulatory authority.

Some national authorities require a limited clinical evaluation for licensingpurposes whenever a new vaccine strain is introduced.

AuthorsThe first draft of these revised Recommendations for Influenza Vaccine (Inacti-vated) was prepared by the following WHO consultants:

Dr R. Dobbelaer, Biological Standardization, Louis Pasteur Scientific Institute ofPublic Health, Brussels, Belgium; Dr R. Levandowski, Center for Biologics Evalua-tion and Research, Food and Drug Administration, Rockville, MD, USA; and Dr J.Wood, National Institute for Biological Standards and Control, Potters Bar, Herts.,England.

A second draft was prepared after an informal WHO Consultation held in Ferney-Voltaire, France (10–11 July 2003), attended by the following participants:

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Dr T. Colegate, Influenza Technical Affairs Manager, Powderject Pharmaceuticals,Liverpool, England; Dr R. Dobbelaer, Head, Biological Standardization, LouisPasteur Scientific Institute of Public Health, Brussels, Belgium; Dr G. Grohmann,Therapeutic Goods Administration, Woden ACT, Australia; Dr A. Hampson, WHOCollaborating Centre for Reference and Research on Influenza, Victoria, Australia;Dr O. Kistner, Director Virology, Baxter BioScience, Baxter Vaccine AG, Orth/Donau, Austria; Dr R. Levandowski, Center for Biologics Evaluation and Research,Food and Drug Administration, Rockville, Rockville, MD, USA; Professor N.V.Medunitsin, Director, Tarasevic State Research Institute for Standardization andControl of Medical Biological Preparations, Moscow, Russian Federation; Dr G.Schild, Emeritus Professor, Imperial College School of Medicine, London, England;Dr J. Shin, Senior Research Scientist, Division of Viral Products, Biological Evalu-ation Department, Korea Food and Drug Administration, Seoul, Republic of Korea,Dr M. Tashiro, Department of Virology III, National Institute of Infectious Diseases,Tokyo, Japan; Dr T. van der Stappen, National Institute of Public Healthand the Environment, Bilthoven, the Netherlands; Dr R. Winsnes, Norwegian Medi-cines Agency, Section for Vaccines, Oslo, Norway; Dr J. Wood, Division of Virol-ogy, National Institute for Biological Standards and Control, Potters Bar, Herts.,England; Dr Zhou Tiequn, Deputy Director, Department of Viral Vaccines, NationalInstitute for the Control of Pharmaceutical and Biological Products, Temple ofHeaven, Beijing, People’s Republic of China.

AcknowledgementsAcknowledgements are due to the following experts for their useful and advice,following comments received on the second draft of these recommendations:

Dr A. Hampson, WHO Collaborating Centre for Reference and Research onInfluenza, Parkville, Victoria, Australia; Dr M. Tashiro, National Institute of InfectiousDiseases, Tokyo, Japan; Dr J. Wood, National Institute for Biological Standardsand Control, Potters Bar, Herts., England.

References1. WHO Expert Committee on Biological Standardization. Twentieth report.

Geneva, World Health Organization, 1968, Annex 2 (WHO Technical ReportSeries, No. 384).

2. WHO Expert Committee on Biological Standardization. Twenty-fifth report.Geneva, World Health Organization, 1973, Annex 1 (WHO Technical ReportSeries, No. 530).

3. WHO Expert Committee on Biological Standardization. Twenty-ninth report.Geneva, World Health Organization, 1978, Annex 3 (WHO Technical ReportSeries, No. 626).

4. WHO Expert Committee on Biological Standardization. Thirtieth report.Geneva, World Health Organization, 1979, Annex 3 (WHO Technical ReportSeries, No. 638).

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5. WHO Expert Committee on Biological Standardization. Forty-first report.Geneva, World Health Organization, 1991, Annex 2 (WHO Technical ReportSeries, No. 814).

6. Brown F et al. Inactivated influenza vaccines prepared in cell culture.Developments in Biological Standardization, 1999, 98:1–214.

7. Prevention and control of influenza: recommendations of the AdvisoryCommittee on Immunization Practices (ACIP). Morbidity and MortalityWeekly Report, 1999, 48:1–28.

8. Wood JM et al. An improved single-radial-immunodiffusion technique forthe assay of influenza haemagglutinin antigen: application for potencydeterminations of inactivated whole virus and subunit vaccines. Journal ofBiological Standardization, 1977, 5:237–247.

9. WHO Expert Committee on Specifications for Pharmaceutical Preparations.Thirty-second report. Geneva. World Health Organization, 1992, Annex 1(WHO Technical Report Series, No. 823).

10. WHO Expert Committee on Biological Standardization. Forty-second report.Geneva, World Health Organization, 1992 (WHO Technical Report Series,No. 822).

11. Biosafety guidelines for personnel engaged in the production of vaccinesand biological products for medicinal use. Geneva, World HealthOrganization, 1995 (WHO/CDS/BVI/95.5).

12. WHO interim biosafety risk assessment. Production of pilot lots ofinactivated influenza vaccines from reassortants derived from avianinfluenza viruses. Geneva, World Health Organization, 2003 (WHO/CDS/CSR/RMD/2003.5).

13. WHO Expert Committee on Biological Standardization. Forty-seventh report,1998. Geneva, World Health Organization, 1998, Annex 6 (WHO TechnicalReport Series, No. 878).

14. WHO Expert Committee on Biological Standardization. Fifty fourth report,2003. Geneva, World Health Organization, 2005, Annex 4 (WHO TechnicalReport Series, No. 927).

15. WHO Expert Committee on Biological Standardization. Twenty-fifth report.Geneva, World Health Organization, 1973, Annex 4 (WHO Technical ReportSeries, No. 530).

16. WHO Expert Committee on Biological Standardization. Fifty-first report.Geneva, World Health Organization, 2002, Annex 1 (WHO Technical ReportSeries, No. 904).

17. WHO Guidelines on Transmissible Spongiform Encephalopathies. Geneva,World Health Organization, 2003 (WHO/BCT/QSD/03.01).

18. WHO Expert Committee on Biological Standardization. Forty-third report.Geneva, World Health Organization, 1994 (WHO Technical Report Series,No. 840).

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Appendix 1Summary protocol for influenza vaccine(inactivated) (master/working seed lot Type A orType B)

The model summary protocol that follows is provided as generalguidance to manufacturers. It is not intended to constrain them inthe presentation of data relevant to the complete review of thequality control tests performed on the vaccine. It is important tonote that satisfactory test results do not necessarily imply that thevaccine is safe and effective, since many other factors must be takeninto account, including the characteristics of the manufacturingfacility.

Name and address of manufacturer

Laboratory reference no. of lot

Date when the processing was completed

Information on manufacture

Virus used to inoculate eggs or cells for the manufacture of the lot:

(a) strain and substrain(b) passage level(c) source and reference no.(d) remarks

Results of sterility test

Results of tests for extraneous agents

Results of tests on adjuvant (if any)

Conditions of storage

Monovalent virus pool Type A or Type B

Name and address of manufacturer

Laboratory reference no. of virus pool

Virus used to inoculate eggs or cells.

(a) master seed strain and source(b) passage level of master seed(c) working seed lot, reference no. and source

Date of inoculation

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Date of harvesting allantoic or amniotic fluids or cell culture fluids

Storage conditions before inactivation

Date of inactivation

Time of inactivation

Method of inactivation

Concentration of inactivating agent

Storage conditions after inactivation

Concentration/purification procedure

Antibiotics used during preparation, if any

Identification of adjuvant added, if any

Tests on monovalent pool1

Test for absence of viable influenza virus

No. of eggs or cell culture vessels inoculated

Incubation time and temperature

Date of test

Results

Determination of haemagglutinin content

Method

Date of determination

Results

Tests for presence of neuraminidase (if performed)

Method

Date of test

Results

Virus disruption (for split vaccine)

Method

Date

Results

1 If there are more than four virus pools in the monovalent pool, the relevant data shouldbe given on a separate sheet.

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Purity (for subunit vaccine)

Method

Date

Results

Purity (for cell-derived vaccine)

Method

Date

Results

Identity tests

Method

Date of test

Results

Test for extraneous agents (if performed)

Method

Date

Results

Final bulk

Name and address of manufacturer

Identification of final bulk

Identification of monovalent virus pool used to prepare final bulk

Date of manufacture

Control of final bulk

Preservative(s) added and concentration

Any other substances added and concentration

Determination of haemagglutinin content

Method

Date of determination

Results

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Sterility

Date of test

Results

Total protein content

Method

Date of test

Results

Ovalbumin content (egg-derived vaccines)

Method

Date of test

Results

Test for residual DNA (if performed)

Method

Date

Results

Test for adjuvant (if performed)

Method

Date

Results

Tests for chemicals used

Date of tests

Results

Final lotIdentity test

Method

Date of test

Results

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Sterility

Method

Date of test

Results

Determination of haemagglutinin content

Method

Date of determination

Results

Innocuity (if performed)

No. and species of animals

Doses injected

Period of observation

Date of test

Results

Endotoxin content

Method

Date of test

Results

Inspection of final container

Results

Other testsAdditional comments (if any)

A sample of a completed final container label and package insertshould be attached.

Certification by producer

Name of head of production of the final vaccine

Certification by head of the quality assurance departmenttaking overall responsibility for production and control of the finalvaccine:

I certify that lot no . . . of influenza vaccine (inactivated), whose num-ber appears on the label of the final container, meets all national

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requirements1 and satisfies Part A of the Requirements for BiologicalSubstances No. 17, revised 1990.

Signature: Name (typed): Date:

Certification by the national controller

If the vaccine is to be exported, provide a copy of the certificate fromthe national regulatory authority as described in section B.2, a label ofa final container, and a leaflet of instructions to users.

1 If any national requirement(s) is (are) not met, specify which one(s) and indicate whyrelease of the lot has nevertheless been authorized.

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Appendix 2Reference laboratories

WHO collaborating centres for reference and researchon influenza

Centers for Disease Control, Atlanta, GA, USA

National Institute for Medical Research, Mill Hill, London, England

National Institute for Infectious Disease, Tokyo, Japan

WHO Collaborating Center for Reference and Research onInfluenza, Melbourne, Australia

Custodian laboratories for candidate influenza vaccineviruses and antigen reagents for vaccine potency

National Institute for Biological Standards and Control, Potters Bar,Herts., England

Center for Biologics Evaluation and Research, Food and DrugAdministration, Bethesda, MD, USA

Therapeutic Goods Administration, Canberra, Australia

National Institute for Infectious Diseases, Tokyo, Japan

Centers for Disease Control, Atlanta, GA, USA

National Institute for Medical Research, Mill Hill, London, England

WHO Collaborating Centre for Reference and Research on Influ-enza, Melbourne, Australia

Laboratories performing calibration of haemagglutinincontent

National Institute for Biological Standards and Control, Potters Bar,Herts., England

Center for Biologics Evaluation and Research, Food and DrugAdministration, Bethesda, MD, USA

Therapeutic Goods Administration, Canberra, Australia

National Institute for Infectious Diseases, Tokyo, Japan

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Appendix 3Model certificate for the release of influenzavaccine (inactivated)1

The following lots of influenza vaccine (inactivated) produced by_________2 in _________3 whose numbers appear on the labels of thefinal containers, meet all national requirements,4 Part A of the Rec-ommendations for Influenza Vaccine (Inactivated) (revised 2003)5

and the recommendatons for good manufacturing practice and qual-ity assurance for biological products.6

Lot Number Date of last Expiry lotpotency test by numbermanufacturer

As a minimum, this certificate is based on examination of the manu-facturing protocol.

The number of this certificate is:

The Director of the National Control Laboratory (or Authority asappropriate):7

Name (typed):

Signature:

Date:

1 To be provided by the national regulatory authority of the country where the vaccineshave been manufactured, on request by the manufacturer.

2 Name of manufacturer.3 Country.4 If any national requirement(s) is (are) not met, specify which one(s) and indicate why

release of the lot(s) has nevertheless been authorized by the national regulatoryauthority.

5 Published in WHO Technical Report Series, No. 927, 2005, Annex 3 and with theexception of the provisions on shipping, which the national regulatory authority may notbe in a position to control.

6 Published in WHO Technical Report Series, No. 822, 1992, Annexes 1 and 2.7 Or his or her representative.

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 4Requirements for the use of animal cells as in vitrosubstrates for the production of biologicals(Addendum 2003)

Introduction

World Health Organization (WHO) Requirements for the use ofanimal cells as in vitro substrates for the production of biologicals (1)provide information on a WHO cell bank of Vero cells. These cellswere developed in 1987 and designated as a Master Cell Bank in 1998.Producers of biologicals and national control authorities can obtaincultures of these Vero cells (free of charge), as well as additionalinformation, from WHO.

At its fifty-third meeting in February 2003, the Expert Committee onBiological Standardization was informed of the outcome of a meetingof a WHO Monitoring Group on Cell Banks held in Potters Bar,England, in October 2002 (2). The Monitoring Group noted thatsignificant changes in regulatory expectations and technological ad-vances had occurred in the requirements of cell bank operation andtesting since the development of the WHO Vero bank 10-87 in 1987and its designation as a Master Cell Bank in 1998. The Committeeendorsed a recommendation from the Monitoring Group that the10-87 bank should not be considered as a Master Cell Bank for directuse in manufacturing processes. Rather, the 10-87 bank should beregarded as a Cell Seed qualified by scientific analytical consensusfrom which Master Cell Banks may be established for thoroughrequalification. The Committee noted (3) that it would be necessaryto revise the Requirements for the use of animal cells as in vitrosubstrates for the production of biologicals (WHO Technical ReportSeries, No. 878, 1998) to accommodate this change.

Manufacturers testing regimes for Master Cell Banks derived fromthe WHO Vero 10-87 Cell Seed will need to extend beyond the testsused to establish the WHO Vero 10-87 bank to include techniquessuch as product enhanced reverse transcriptase (PERT) assays (2).Furthermore, manufacturers should be continually aware of currentdevelopments regarding adventitious agents and ensure that data

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from safety testing on banks of cells used in manufacturing processesare regularly reviewed and updated where appropriate.

The redesignation of the WHO Vero cell bank 10-87 as a Cell Seedmay lead to investigations into the use of cells for manufacturingprocesses at higher population doublings than have previously beenrecommended. The potential for increased tumorigenicity at higherpopulation doublings, among other issues, must therefore be consid-ered (2). This assessment of tumorigenicity should take into accountthe variation that may occur in assessment of population doublings(both between and within laboratories), the potential for variability ofin vivo tumorigenicity tests and the variation between different cell-culture processes. The establishment of arbitrary passage limits forthe use of cells in a manufacturing process may be less important thancareful process validation and testing of cells passaged beyond theprocess limits.

The amendments to the 1998 Requirements apply to the generalconsiderations section and are listed below.

General considerationsContinuous-cell-line substrates

The last paragraph on page 23 of WHO Technical Report Series No.878 currently reads:

“The WHO master cell bank of Vero cells is stored at the EuropeanCollection of Animal Cell Cultures (ECACC), Porton Down, En-gland and the American Type Culture Collection (ATCC), Rockville,MD, USA. Producers of biologicals and national control authoritiescan obtain cultures of these Vero cells (free of charge), as well asadditional background information, from Biologicals, World HealthOrganization, 1211 Geneva 27, Switzerland.”

This paragraph should be replaced by the following:

“The WHO 10-87 cell bank of Vero cells is stored at the EuropeanCollection of Animal Cell Cultures (ECACC), Porton Down, En-gland and the American Type Culture Collection (ATCC), Rockville,MD, USA. This cell bank should be regarded as a Cell Seed qualifiedby scientific analytical consensus from which Master Cell Banks maybe established for thorough re-qualification. Producers of biologicalsand national regulatory authorities can obtain cultures of these Verocells (free of charge), as well as additional background information,from Quality Assurance and Safety of Biologicals, World HealthOrganization, 1211 Geneva 27, Switzerland.”

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References1. Requirements for the use of animal cells as in vitro substrates for the

production of biologicals. In: WHO Expert Committee on BiologicalStandardization. Forty-seventh report. Geneva, World Health Organization,1998, Annex 1 (WHO Technical Report Series, No. 878).

2. Report of the WHO Monitoring Group on Cell Banks, 16-17 October 2002.

3. WHO Expert Committee on Biological Standardization. Fifty-second report.Geneva, World Health Organization, 2004 (WHO Technical Report Series, No.924, page 15).

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 5Recommendations for diphtheria, tetanus, pertussisand combined vaccines (Amendments 2003)

Introduction

These amendments should be read in conjunction with the introduc-tion to the Requirements for diphtheria, tetanus, pertussis and com-bined vaccines published in WHO Technical Report Series, No. 800,1990 (Annex 2).

Diphtheria and tetanus vaccines are among the most frequently usedvaccines worldwide and have been remarkably successful products.Their use has resulted in a significant decrease in the incidenceof these diseases in both the industrialized world and in developingcountries. Nevertheless, some difficulties exist in the global har-monization of potency testing procedures, even when InternationalStandards are used, and different approaches have been taken bydifferent countries. Some follow WHO and European Pharmaco-poeia procedures, whereas others follow the National Institutes ofHealth (NIH) procedures used in the USA, with or withoutmodifications.

The approach taken by the European Pharmacopoeia, like that ofWHO, is based on the determination of the immunizing potency ofeach final bulk by comparison with an appropriate reference materialcalibrated against the International Standard for Diphtheria Toxoid(adsorbed) or the International Standard for Tetanus Toxoid(adsorbed), as appropriate (1, 2). There has been much activity inrecent years aimed at simplifying the current tests, reducing the num-ber of animals used and refining the end-point used in potency testing.Some studies have also considered the possibility of using the sameanimals to test the potency of several antigens.

The approach taken by the USA is based on the NIH assays (3–6)where the minimal acceptable potency is defined as the capacity of atest vaccine to induce an antibody response that reaches or surpassesthe threshold of 2 units per ml. A suitable reference antitoxin, towhich “units/ml” have been assigned, is used to express antibodyconcentration in relative terms, as measured by an in vivo toxinneutralization assay.

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The inclusion of a control vaccine in the NIH test is being consideredand would, in principle, improve control of the variations in theimmune response induced in animals. The application of the Vero cellassay for the detection of anti-diphtheria toxin neutralizing antibodiesis also being considered in the USA. Also, the expression of antibodylevels in International Units could be achieved by calibration of thereference antitoxin against the International Standard for antiserum(see section A.1.3).

Despite many attempts to harmonize potency requirements globally,there are still no universally accepted methods. This leads toproblems in international exchange of these vaccines arisingfrom difficulties in the mutual recognition of the results of testing.The development of new combination vaccines, has led to an in-creased need for harmonization of the diphtheria and tetanus potencytests, creating a unique opportunity to resolve this long-standingissue.

The purpose of the potency test is to assess in a suitable animal modelthe capacity of the product being tested to induce a protective re-sponse analogous to that of toxoids shown to be efficacious in hu-mans. The potency test has two stages. During the first stage aprotective response is induced in mice or guinea-pigs, and during thesecond stage the protective response is measured by direct or indirectmethods.

Considerable international consultation has identified the need toclarify the current WHO text relating to the introduction and use ofsimplified potency assays for the purpose of routine lot release. Thisshould be seen as a first step towards the revision of the whole text ofthe current WHO Requirements (Recommendations) for Diphtheria,Tetanus, Pertussis and Combined Vaccines. The following amend-ments have thus been made to Annex 2, WHO Technical ReportSeries, No. 800, 1990. These include:

1. The updating of sections on International Reference Prepara-tions for Diphtheria vaccine (adsorbed) and Tetanus vaccine(adsorbed);

2. The division of the sections on potency for Diphtheria vaccine(adsorbed) and for Tetanus vaccine (adsorbed) into two subsec-tions to clearly distinguish the recommendations for licensing fromthose for routine batch release;

3. Simplification of the routine testing for batch release and use offewer animals than used for licensing;

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4. Amendment of the recommendations for diphtheria and tetanuspotency testing in the diphtheria, tetanus, pertussis combinedvaccine section to bring them in line with the changes outlined(in 2 and 3) above.

No changes have yet been made to the pertussis section of the Re-quirements for pertussis vaccines published in Technical ReportSeries, No 800, 1990 (annex 2).

Requirements for diphtheria vaccine (adsorbed)

Part A. Manufacturing recommendationsReplace section A.1.3, International reference materials, by thefollowing:

A.1.3 International reference materialsThe first International Reference Reagent of Diphtheria Toxoid forFlocculation Tests was established in 1988 (7).

The Third International Standard of Diphtheria Toxoid Adsorbedwas established in 1999 (8) for determining the potency of vaccinescontaining diphtheria toxoid. The assigned activity of 160IU/ampouleis based on its calibration in guinea-pig challenge assays. Potenciescalculated by other methods should not be assumed to be transferablewithout validation. When potency tests are carried out in mice insteadof guinea-pigs, transferability should be demonstrated.

The International Standard for Diphtheria Antitoxin1 was establishedin 1934. It is made from horse hyperimmune serum for use in toxinneutralization potency assays, in vivo.

The above-mentioned reference materials are in the custody of theNational Institute for Biological Standards and Control, Potters Bar,Herts., England (web site: http://www.nibsc.ac.uk). The WHO cata-logue of international biological standards should be consulted for thelatest list of appropriate international standards and reference mate-rials (http://www.who.int/biologicals). International reference materi-als are intended for the calibration of national reference materials foruse in the manufacture and laboratory control of diphtheria antitoxinand vaccines.

1 The original standard is a freeze-dried preparation and new standard is a liquid fill of10 IU/ml, made every 2 years.

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Replace section A.3.5.6, Potency, by the following:

A.3.5.6 Potency

a) Potency test for licensingThe potency of the final bulk is determined by comparison with anappropriate reference material1 calibrated against the InternationalStandard for Diphtheria Toxoid, Adsorbed. A three-dilution assayshould be used to evaluate consistency of production of the vaccine inquestion. Three-dilution assays should also be used to test productstability for the purpose of establishing shelf-life as well as to calibratereference preparations.

Potency should be determined by the inoculation of guinea-pigs withappropriate doses or dilutions of both the tested product and thereference material. After immunization, guinea-pigs may be chal-lenged either by the subcutaneous or the intradermal route, or bled toobtain sera for measurement of the antitoxin or antibody response.When guinea-pigs are bled, the antibody levels of the individual ani-mals may be titrated by means of toxin neutralization tests in vivo orin vitro, such as the Vero cell assay.

The ELISA assay (9) or another suitable in vitro method may be used tomeasure the antibody response to diphtheria toxoid provided these assayshave been validated against the challenge assay or the toxin neutralizationtest, using the particular product in question. A minimum of three assayswith a suitable dose–response range is likely to be required for validation.

Appropriate statistical methods should be used to calculate the po-tency of the final bulk (9). The national regulatory authority shouldapprove the method and the interpretation of the results.

If mice are used for the potency assay, they should be bled andantibody levels of the individual animals titrated by means of toxinneutralization tests in vivo in guinea-pigs, or in vitro using the Verocell assay. Because mice are not sensitive to diphtheria toxin, chal-lenge with diphtheria toxin is not possible.

The ELISA or toxoid-binding inhibition (ToBI) assay (9) or another suitablemethod may be used to measure the antibody response to diphtheriatoxoid, provided these assays have been validated against the toxinneutralization test, using the particular product in question. A minimum ofthree assays with a suitable dose–response range is likely to be required forvalidation.

The potency of diphtheria vaccine used for the immunization of chil-dren should not be less than 30IU per single human dose. The results

1 Such material could be monocomponent or multicomponent.

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of all statistically valid tests should be combined in a geometric meanestimate and the confidence limits calculated. If the lower limit of the95% confidence interval of the estimated potency is less than 30IUper single human dose, then the limits of the 95% confidence intervalshould be within 50–200% of the estimated potency.

The potency values mentioned above do not apply to diphtheriavaccine for use in adolescents or adults.

b) Potency test for routine lot releaseFollowing licensing, and once consistency in production and qualitycontrol of the vaccine has been further confirmed on a continuousbasis, then the determination of potency in routine lot release may,with the approval of the national regulatory authority, be based onthe results of serological assays, or on a challenge assay, both involv-ing a reduced number of animals and/or doses.

To further confirm consistency on a continuous basis, the potency ofabout ten recent batches of vaccine should be tested using the fullthree-dilution assay. If potency expressed in International Units isrelatively uniform and if the expectations of linearity and parallelismare consistently satisfied, then fewer doses may be used and theassumptions of linearity and parallelism need not be tested in eachassay. When vaccine lots consistently give a lower limit of the 95%confidence intervals for the estimated potency well in excess of 30IUper single human dose, one-dilution tests may offer advantages. Ifone-dilution assays are not advantageous, a reduction in animal usagemay, nevertheless, be achieved by use of two-dilution assays oranother suitable design modification.

A one-dilution assay is based on the same principles for evaluatingthe response as the three-dilution assays. The assay involves the selectionof a dose of the reference vaccine, expressed as a fraction of 30IU (i.e. ofthe minimum potency of a single human dose), that elicits a minimumprotective effect in guinea-pigs, and comparing its effect with the responseelicited by the same fraction of a human dose of the test vaccine. Ifthe response to the test vaccine is significantly greater than the responseto the reference vaccine (P £ 0.05), the potency of the test vaccine issatisfactory.

One-dilution assays provide assurance that the lower limit of theestimated potency is in excess of the minimum requirement. A disad-vantage of such an approach is that strictly quantitative estimates ofvaccine potency will not be possible.

If in vitro serological assays are used, they should show that theproduct induces an appropriate antibody response in animals in com-parison with a reference material calibrated against the InternationalStandard for Diphtheria Toxoid, Adsorbed.

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The ELISA assay (9) or another suitable in vitro method may be used tomeasure the antibody response to diphtheria toxoid, provided these assayshave been validated against the challenge assay or the toxin neutralizationtest, using the particular product in question. A minimum of three assayswith a suitable dose–response range is likely to be required for validation ofa particular product in a particular laboratory. These methods will requireprecise definition of the characteristics of reagents critical for successfulperformance of the testing method which may include positive and negativecontrol sera, antigen and others.

There is a need to support the data generated by a simplified potencyassay with physicochemical methods to ensure overall consistency ofproduction.

Lot release based on a simplified approach will require periodicreview to ensure that the validity of all procedures is maintained.The timing of the review should be decided on a case-by-case basisdepending on the number of batches of vaccine produced annuallyand/or by time (at least every 2 years), as agreed by the nationalregulatory authority.

Recommendations for tetanus vaccine (adsorbed)

Part A. Manufacturing recommendationsReplace section A.1.3, International reference materials, by thefollowing:

A.1.3 International reference materialsThe first International Reference Reagent of Tetanus Toxoid forFlocculation Tests was established in 1988 (7).

The third International Standard of Tetanus Toxoid, Adsorbed,was established in 2000 (10) for determining the potency of vaccinescontaining tetanus toxoid. The assigned value of 469IU/ampouleis based on its calibration in guinea-pig challenge assays. Potenciescalculated by other methods should not be assumed to be transferablewithout validation. When potency tests are carried out in mice insteadof guinea-pigs, transferability should be demonstrated.

The first International Standard for Tetanus Immunoglobulin,human was established in 1992 (11) for use in toxin neutralizationpotency tests.

The above-mentioned international standards are in the custodyof the National Institute for Biological Standards and Control,Potters Bar, Hertfordshire, EN6 3QG. England (web site: http://www.nibsc.ac.uk). The WHO catalogue of international biologicalstandards should be consulted for the latest list of appropriateinternational standards and reference materials (http://www.who.int/

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biologicals). The international reference materials are intended forthe calibration of national reference materials for use in the manufac-ture and laboratory control of tetanus antitoxin and vaccines.

Replace section A.3.5.6, Potency, by the following:

A.3.5.6 Potencya) Potency test for licensing

The potency of the final bulk should be determined by comparisonwith an appropriate reference material1 calibrated against the Inter-national Standard for Tetanus Toxoid, Adsorbed. A three-dilutionassay should be used to evaluate consistency of production of thevaccine in question. Three-dilution assays should also be used to testproduct stability for the purpose of establishing shelf-life as well as tocalibrate reference preparations.

The potency should be determined by the inoculation of guinea-pigsor mice with appropriate doses or dilutions of both the tested productand the reference material. After immunization, animals may be chal-lenged by the subcutaneous route, or bled to obtain sera for measure-ment of the antitoxin response. When animals are bled, the antibodylevels of the individual animals may be titrated by means of toxinneutralization tests in vivo.

The ELISA or ToBI assay (9) or another suitable method may be used tomeasure the antibody response to tetanus toxoid, provided these assayshave been validated against the challenge assay or the toxin neutralizationtest, using the particular product in question. A minimum of three assayswith a suitable dose-response range is likely to be required for validation.

Appropriate statistical methods should be used to calculate the po-tency of the final bulk (9). The national regulatory authority shouldapprove the method and the interpretation of the results.

The potency of tetanus vaccine used for the immunization of childrenshould not be less than 40IU per single human dose. The results of allstatistically valid tests must be combined in a geometric mean esti-mate and its confidence limits should be calculated. If the lower limitof the 95% confidence interval of the estimated potency is less than40 IU per single human dose, then the limits of the 95% confidenceinterval should be within 50–200% of the estimated potency.

In some countries these potency values may not apply to tetanus vaccinefor adolescent or adult use.2

1 Such material could be monocomponent or multicomponent.2 Further guidance will be developed.

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b) Potency test for routine lot releaseFollowing licensing, and once consistency in production and qualitycontrol of the vaccine has been further confirmed on a continuousbasis, then the determination of potency in routine lot release may,with the approval of the national regulatory authority, be based onthe results of serological assays, or on a challenge assay, both involv-ing a reduced number of animals and/or doses.

To further confirm consistency on a continuous basis, the potency ofabout ten recent batches of vaccine should be tested using the fullthree-dilution assay. If potency expressed in International Units isrelatively uniform and if the expectations of linearity and parallelismare consistently satisfied, then fewer doses may be used and theassumptions of linearity and parallelism need not be tested in eachassay. When vaccine potencies consistently give a lower limit of the95% confidence intervals for the estimated potency in excess of 40IUper single human dose, one-dilution tests may offer advantages.If one-dilution assays are not advantageous, a reduction in animalusage may, nevertheless, be achieved by use of two-dilution assays oranother suitable design modification.

A one-dilution assay is based on the same principles for evaluating theresponse as the three-dilution assays. The assay involves the selection of adose of the reference vaccine, expressed as a fraction of 40 IU (i.e. of theminimum potency of a single human dose), that elicits a minimal protectiveeffect, and comparing its effect with the response elicited by the samefraction of a human dose of the test vaccine. If the response to the testvaccine is significantly greater than the response to the reference vaccine(P £ 0.05), the potency of the test vaccine is satisfactory.

One-dilution assays provide assurance that the lower limit of theestimated potency is in excess of the minimum requirement. A disad-vantage of such an approach is that strictly quantitative estimates ofvaccine potency cannot be obtained.

In vitro serological assays should show that the product inducesan appropriate antibody response in animals in comparison with areference material calibrated against the International Standard forTetanus Toxoid, Adsorbed.

The ELISA or ToBI assay (9) or another suitable method may be used tomeasure the antibody response to tetanus toxoid, provided these assayshave been validated against the challenge assay or the toxin neutralizationtest, using the particular product in question. A minimum of three assayswith a suitable dose–response range is likely to be required for validation fora particular product in a particular laboratory. These methods will requireprecise definition of the characteristics of reagents critical for successfulperformance of the testing method which may include positive and negativecontrol sera, antigen and others.

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There is a need to support the data generated by a simplified potencyassay with physicochemical methods to ensure overall consistency ofproduction.

Lot release based on a simplified approach will require periodicreview to ensure that validity of all procedures is maintained. Thetiming of the review should be decided on a case-by-case basis de-pending on the number of batches of vaccine produced annually, orby time (e.g. every 2 years), as agreed by the national regulatoryauthority.

Recommendations for combined vaccines(adsorbed)

Part A. Manufacturing recommendationsA.2 Special tests for DTP vaccinesA.2.1 Final bulkReplace section A.2.1.1, Potency test, by the following:

The following tests should be carried out on the final bulk vaccine.

A.2.1.1 Potency test

For the Diphtheria component, the recommendations for the licens-ing and routine lot release of Diphtheria vaccine (adsorbed) shouldapply (section A.3.5.6).

For the Tetanus component, the potency of which is tested in guinea-pigs, the recommendations for licensing and for routine lot release ofTetanus vaccine (adsorbed) should apply (section A.3.5.6). However,when tetanus toxoid is in combination with whole-cell pertussis vac-cine and when the potency test for licensing is performed in mice, theestimated potency of tetanus vaccine used for immunization of chil-dren should be not less than 60IU per single human dose. The samepotency criteria should also apply when carrying out the routine lotrelease test.

AuthorsThe first draft of this amendment was prepared at a WHO Informal Consultation(meeting of drafting group) held in Geneva, 30 June–2 July 2003 and attended bythe following participants: Dr J. Arciniega, Office of Vaccines Review andResearch, Center for Biologics Evaluation and Research, Bethesda, MD, USA; DrM. Corbel, Division of Bacteriology, National Institute for Biological Standards andControl, Potters Bar, Herts., England; Dr R. Gaines Das, Statistics Department,National Institute for Biological Standards and Control, Potters Bar, Herts.,

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England; Dr D. Sesardic, Division of Bacteriology, National Institute for BiologicalStandards and Control, Potters Bar, Herts., England, Dr E. Griffiths, Biologics andGenetic Therapies, Ottawa, Canada; Dr S. Jadhav, Serum Institute of India, Pune,India; Dr J.G. Kreeftenberg, International Support, Netherlands Vaccine Institute,Bilthoven, the Netherlands, and Dr I. Knezevic, Quality Assurance and Safety:Biologicals, World Health Organization, Geneva, Switzerland.

The final draft was prepared by the drafting group, taking into account commentsmade by the reviewers of the document.

References1. Assay of diphtheria vaccine adsorbed (2.7.6); Method of analysis. European

Pharmacopoeia, 4th ed. Strasbourg, Council of Europe, 2003: Supplement7:2630–2632.

2. Assay of tetanus vaccine adsorbed (2.7.8), Method of analysis. EuropeanPharmacopoeia, 4th ed. Council of Europe, Strasbourg, 2003: Supplement7:4257–4260.

3. US Department of Health, Education and Welfare Public Health Service,National Institute of Health. Minimum requirements: tetanus toxoid. Fourthrevision. Bethesda, MD, NIH, 1952.

4. US Department of Health, Education and Welfare Public Health Service,National Institute of Health. Minimum Requirements: Diphtheria Toxoid.Fourth revision. Bethesda, MD, NIH, 1947.

5. US Department of Health, Education and Welfare Public Health Service,National Institute of Health. Minimum Requirements: Tetanus and DiphtheriaToxoids Combined Precipitated Adsorbed (For Adult Use). Bethesda, MD,NIH, 1953.

6. US Department of Health, Education and Welfare Public Health Service,National Institute of Health. Minimum Requirements: Tetanus and DiphtheriaToxoids Combined Precipitated Adsorbed (For Adult Use). Amendment 1.Bethesda, MD, NIH, 1956.

7. WHO Expert Committee on Biological Standardization. Thirty-ninth report.Geneva, World Health Organization, 1989 (WHO Technical Report Series,No. 786).

8. WHO Expert Committee on Biological Standardization. Fiftieth report.Geneva, World Health Organization, 2002 (Technical Report Series,No. 904), Annex 5.

9. WHO Manual of laboratory methods for testing of vaccines used in the WHOExpanded Programme on Immunization. Geneva, World Health Organization(WHO/VSQ/97.04).

10. WHO Expert Committee on Biological Standardization. Fifty-first report.Geneva, World Health Organization, 2002 (WHO Technical Report Series,No. 910 ).

11. WHO Expert Committee on Biological Standardization. Fiftieth report.Geneva, World Health Organization, 1994 (WHO Technical Report Series,No. 840).

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 6Biological substances: international standards andreference reagents

A list of International Biological Reference Preparations was issuedin WHO Technical Report Series, No. 897, 2000 (Annex 4) and isavailable on the Internet at http://www.who.int/biologicals. Copies ofthe list may be obtained from appointed sales agents for WHO pub-lications or from: Distribution and Sales, World Health Organization,1211 Geneva 27, Switzerland.

The Expert Committee made the following changes to the previouslist.

Additions

Antibodies

Anti-toxoplasma IgG, 20 IU/ampoule First Internationalhuman Standard 2003

This substance is held and distributed by the International Labora-tory for Biological Standards, National Institute for Biological Stan-dards and Control, Potters Bar, Herts. EN6 3QG, England.

Antigens and related substances

Yellow fever vaccine 104.5 IU/ampoule First InternationalStandard 2003

Pertussis toxin 10 000IU/ampoule First InternationalStandard 2003

These substances are held and distributed by the International Labo-ratory for Biological Standards, National Institute for BiologicalStandards and Control, Potters Bar, Herts. EN6 3QG, England.

Blood products and related substances

Factor VIII, concentrate, 11.0 IU/ampoule Seventh Internationalplasma, human Standard 2003

Factor VIII and von 0.68IU/ampoule factor Fifth International

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Willebrand factor, VIII:C Standard 2003plasma, human 0.94IU/ampoule factor

VIII:antigen0.91IU/ampoule vonWillebrand factor:antigen0.78IU/ampoulevon Willebrandfactor:ristocetincofactor0.94IU/ampoule vonWillebrandfactor:collagenbinding

Prekallikrein activator, 29 IU/ampoule Second Internationalhuman Standard 2003

Low-molecular-weight 1097IU/ampoule Second Internationalheparin Anti-Xa Standard 2003

326IU/ampouleAnti-Iia

These substances are held and distributed by the InternationalLaboratory for Biological Standards, National Institute forBiological Standards and Control, Potters Bar, Herts. EN6 3QG,England.

Cytokines, growth factors and endocrinological substancesInterferon, beta, human, 40 000 IU/ampoule Third Internationalrecombinant, Standard 2003glycosylated

Tumour necrosis factor, 46 500 IU/ampoule Second Internationalalpha, human, Standard 2003recombinant

Luteinizing hormone, 189 IU/ampoule First Internationalhuman recombinant Standard 2003

Thyroid-stimulating 11.5 ¥ 10-3 IU/ampoule Third Internationalhormone, human, for Standard 2003immunoassay

These substances are held and distributed by the InternationalLaboratory for Biological Standards, National Institute forBiological Standards and Control, Potters Bar, Herts. EN6 3QG,England.

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Diagnostic reagents

Hepatitis B surface 33 IU/vial Second Internationalantigen Standard 2003Hepatitis B surface No assignment First Internationalantigen panel (set of 4 Reference Panel 2003dilutions and control)

These substances are held and distributed by the InternationalLaboratory for Biological Standards, National Institute for BiologicalStandards and Control, Potters Bar, Herts. EN6 3QG, England.

Lipoprotein (a) for 0.107 nanomoles/vial First Referenceimmunoassay Reagent 2003

This substance is held and distributed by Northwest Lipid ResearchLaboratories, University of Washington School of Medicine, 2121North 35th Street, Seattle, WA 98103, USA.

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© World Health OrganizationWHO Technical Report Series, No. 927, 2005

Annex 7Recommendations and guidelines for biologicalsubstances used in medicine and other documents

The recommendations (previously called requirements) and guide-lines published by the World Health Organization are scientific andadvisory in nature but may be adopted by a national regulatory author-ity as national requirements or used as the basis of such requirements.

These international recommendations are intended to provide guid-ance to those responsible for the production of biologicals as well asto others who may have to decide upon appropriate methods of assayand control in order to ensure that these products are safe, reliableand potent.

Recommendations concerned with biological substances used inmedicine are formulated by international groups of experts and arepublished in the Technical Report Series of the World Health Orga-nization,1 as listed here. A historical list of requirements and othersets of recommendations is available on request from the WorldHealth Organization, 1211 Geneva 27, Switzerland.

Reports of the Expert Committee on Biological Standardization pub-lished in the WHO Technical Report Series can be purchased from:

Marketing and DisseminationWorld Health Organization1211 Geneva 27SwitzerlandTelephone: +41 22 79 12 476Fax: +41 22 79 14 857email: publications@who.int

Individual recommendations and guidelines may be obtained free ofcharge as offprints by writing to:

Quality Assurance and Safety of BiologicalsDepartment of Immunization, Vaccines and BiologicalsWorld Health Organization1211 Geneva 27

Switzerland

1 Abbreviated in the following pages as TRS.

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Recommendations, guidelines and other documentsRecommendations and Guidelines Reference

Acellular pertussis component of monovalent or Adopted 1996, TRS 878 (1998)combined vaccines

Animal Cells, use of, as in vitro Substrates for the Revised 1996, TRS 878 (1998);Production of Biologicals Addendum 2003, TRS 927 (2005)

BCG Vaccine, dried Revised 1985, TRS 745 (1987);Amendment 1987, TRS 771 (1988)

Biological products prepared by recombinant DNA Adopted 1990, TRS 814 (1991)technology

Blood, Blood Components and Plasma Derivatives: Revised 1992, TRS 840 (1994)collection, processing and quality control

Blood plasma products, human: viral inactivation Adopted 2001, TRS 924 (2002)and removal procedures

Cholera Vaccine (Inactivated, oral) Adopted 2001, TRS 924 (2002)

Diphtheria, Tetanus, Pertussis and Combined Revised 1989, TRS 800 (1990);Vaccines Ammendment 2003, TRS 927 (2005)

DNA Vaccines Adopted 1996, TRS 878 (1998)

Haemophilus influenzae Type b Conjugate Vaccines Revised 1998, TRS 897 (2000)

Haemorrhagic Fever with Renal Syndrome (HFRS) Adopted 1993, TRS 848 (1994)Vaccine (Inactivated)

Hepatitis A vaccine (inactivated) Adopted 1994, TRS 858 (1995)

Hepatitis B Vaccine prepared from Plasma Revised 1987, TRS 771 (1988)

Hepatitis B Vaccines made by Recombinant DNA Adopted 1988, TRS 786 (1989);Techniques Amendment 1997, TRS 889 (1999)

Human Interferons made by Recombinant DNA Adopted 1987, TRS 771 (1988)Techniques

Human Interferons prepared from Lymphoblastoid Adopted 1988, TRS 786 (1989)Cells

Influenza Vaccine (Inactivated) Revised 2003, TRS 927 (2005)

Influenza Vaccine (Live) Adopted 1978, TRS 638 (1979)

Japanese Encephalitis Vaccine (Inactivated) for Adopted 1987, TRS 771 (1988)Human Use

Japanese Encephalitis Vaccine (Live) for Human Adopted 2000, TRS 910 (2002)Use

Louse-borne Human Typhus Vaccine (Live) Adopted 1982, TRS 687 (1983)

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Recommendations and Guidelines Reference

Measles, Mumps and Rubella Vaccines and Adopted 1992 TRS 848 (1994);Combined Vaccine (Live) Note TRS 848 (1994)

Meningococcal Polysaccharide Vaccine Adopted 1975, TRS 594 (1976);Addendum 1980, TRS 658 (1981);Amendment 1999, TRS 904 (2002)

Meningococcal C conjugate vaccines Adopted 2001, TRS 924 (2002);Addendum 2003, TRS, 926 (2004)

Monoclonal Antibodies Adopted 1991, TRS 822 (1992)

Pneumococcal conjugate vaccines Adopted 2003, 927 (2005)

Poliomyelitis Vaccine (Inactivated) Revised 2000, TRS 910 (2002);Amendment 2003, TRS 926 (2004)

Poliomyelitis Vaccine, Oral Revised 1999, TRS 904 (2002);Addendum 2000, TRS 910 (2002)

Rabies Vaccine (inactivated) for Human Use, Adopted 1986, TRS 760 (1987);Produced in Continuous Cell Lines Amendment 1992, TRS 840 (1994)

Rabies Vaccine for Human Use Revised 1980, TRS 658 (1981);Amendment 1992, TRS 840 (1994)

Rift Valley Fever Vaccine Adopted 1981, TRS 673 (1982)

Smallpox Vaccine Revised 2003, TRS 926 (2004)

Sterility of Biological Substances Revised 1973, TRS 530 (1973);Amendment 1995, TRS 872 (1998)

Synthetic Peptide Vaccines Adopted 1997, TRS 889 (1999)

Thiomersal for vaccines: regulatory expectations Adopted 2003, TRS 926 (2004)for elimination, reduction or removal

Thromboplastins and Plasma Used to Control Revised 1997, TRS 889 (1999)Oral Anticoagulant Therapy

Tick-borne Encephalitis Vaccine (Inactivated) Adopted 1997, TRS 889 (1999)

Tuberculins Revised 1985, TRS 745 (1987)

Typhoid Vaccine Adopted 1966, TRS 361 (1967)

Vaccines, Clinical Evaluation: regulatory Adopted 2001, TRS 924 (2004)expectations

Vaccines, nonclinical evaluation Adopted 2003, TRS 927 (2005)

Varicella Vaccine (Live) Revised 1993, TRS 848 (1994)

Vi Polysaccharide Typhoid Vaccine Adopted 1992, TRS 840 (1994)

Yellow Fever Vaccine Revised 1995, TRS 872 (1998)

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Other documents Reference

Biological standardization and control: a scientific Unpublished documentreview commissioned by the UK National WHO/BLG/97.1Biological Standards Board (1997)

Development of national assay services for TRS 565 (1975)hormones and other substances in communityhealth care

Good manufacturing practices for biological TRS 822 (1992)products

Guidelines for national authorities on quality TRS 822 (1992)assurance for biological products

Guidelines for the preparation, characterization TRS 800 (1990)and establishment of international and otherstandards andreference reagents for biologicalsubstances

Guidelines for quality assessment of antitumour TRS 658 (1981)antibiotics

Guidelines for the safe production and quality Adopted 2003,control of inactivated poliovirus manufactured TRS 926 (2004)from wildpolioviruses

Guidelines on Transmissible Spongiform Unpublished documentEncaphalopathies in relation to biological and WHO/BCT/QSD/03.01pharmaceutical products

Laboratories approved by WHO for the TRS 872 (1998)production of yellow fever vaccine, revised 1995

Production and testing of WHO yellow fever virus TRS 745 (1987)primary seed lot 213-77 and reference batch 168-73

Recommendations for the assessment of binding-assayTRS 565 (1975)systems (including immunoassay and receptor assaysystems) for human hormones and their bindingproteins. (A guide to the formulation of requirementsfor reagents and assay kits for the above assays andnotes on cytochemical bioassay systems.)

Regulation and licensing of biological products in TRS 858 (1987)countries with newly developing regulatoryauthorities

Report on the standardization and calibration of TRS 889 (1997)cytokine immunoassays

Standardization of interferons (reports of WHO TRS 687 (1983)Informal Consultations) TRS 725 (1985)

TRS 771 (1988)

Summary protocol for the batch release of virusvaccines TRS 822 (1992)

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